Cyclodextrin-based polymers for therapeutics delivery

ABSTRACT

The present invention relates to novel compositions of therapeutic cyclodextrin containing polymeric compounds designed as a carrier for small molecule therapeutics delivery and pharmaceutical compositions thereof. These cyclodextrin-containing polymers improve drug stability and solubility, and reduce toxicity of the small molecule therapeutic when used in vivo. Furthermore, by selecting from a variety of linker groups and targeting ligands the polymers present methods for controlled delivery of the therapeutic agents. The invention also relates to methods of treating subjects with the therapeutic compositions described herein. The invention further relates to methods for conducting pharmaceutical business comprising manufacturing, licensing, or distributing kits containing or relating to the polymeric compounds described herein.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/553,400, filedJul. 19, 2012, which is a continuation of U.S. Ser. No. 13/421,839,filed Mar. 15, 2012, now U.S. Pat. No.: 8,252,276 issued Jul. 8, 2012,which is a continuation of U.S. Ser. No. 13/277,780, filed Oct. 20,2011, now U.S. Pat. No.: 8,314,230 issued Nov. 11, 2012, which is adivisional of U.S. Ser. No. 11/881,325, filed Jul. 25, 2007, now U.S.Pat. No.: 8,110,179 issued Jul. 2, 2012, which is a continuation of U.S.Ser. No. 10/656,838 filed Sep. 5, 2003, now U.S. Pat. No. 7,270,808which claims the benefit of U.S. Provisional Patent Application Ser.Nos. 60/408,855, filed on Sep. 6, 2002, 60/422,830 filed Oct. 31, 2002,and 60/451,998 filed on Mar. 4, 2003. The specifications of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Drug delivery of some small molecule therapeutic agents, such ascamptothecin, has been problematic due to their poor pharmacologicalprofiles. These therapeutic agents often have low aqueous solubility,their bioactive forms exist in equilibrium with an inactive form, orhigh systemic concentrations of the agents lead to toxic side-effects.Some approaches to circumvent the problem of their delivery have been toconjugate the agent directly to a water-soluble polymer such ashydroxypropyl methacrylate (HPMA), polyethyleneglycol, andpoly-L-glutamic acid. In some cases, such conjugates have beensuccessful in solubilizing or stabilizing the bioactive form of thetherapeutic agent, or achieving a sustained release formulation whichcircumvents complications associated with high systemic concentrationsof the agent.

Another approach to the drug delivery problem has been to formhost/guest inclusion complexes between the therapeutic agent andcyclodextrins or derivatives thereof. Cyclodextrins (α, β, γ) and theiroxidized forms have unique physico-chemical properties such as goodwater solubility, low toxicity and low immune response. To date, most ofthe drug delivery studies with cyclodextrins have focused on theirability to form supra-molecular complexes, wherein cyclodextrins formhost/guest inclusion complexes with therapeutic molecules and thus alterthe physical, chemical, and/or biological properties of these guestmolecules.

U.S. Pat. No. 5,276,088 describes a method for synthesizingcyclodextrin-containing polymers by either reacting polyvinyl alcohol orcellulose or derivatives thereof with cyclodextrin derivatives, or bycopolymerization of a cyclodextrin derivative with vinyl acetate ormethyl methacrylate.

U.S. Pat. No. 5,855,900 describes a biodegradablecyclodextrin-containing polymer. The patent discloses asupramolecular-structured biodegradable polymeric assembly comprising aplurality of drug-modified α, β, γ-cyclodextrins and a linear polymericchain threading through the structural cavity of the cyclodextrins.

There is an ongoing need for new approaches to the delivery of smalltherapeutic agents that have poor pharmacological profiles such ascamptothecin, paclitaxel, doxorubicin, and cyclosporine A.

SUMMARY OF THE INVENTION

The present invention relates to novel compositions of polymerconjugates, defined as polymeric materials covalently coupled totherapeutic/bioactive agents, as carriers for therapeutics delivery. Inone aspect, the present invention provides water-soluble, biocompatiblepolymer conjugates comprising a water-soluble, biocompatible polymercovalently attached to bioactive moieties through attachments that arecleaved under biological conditions to release the bioactive moieties.In certain such embodiments, the polymer comprises cyclic moietiesalternating with linker moieties that connect the cyclic structures,e.g., into linear or branched polymers, preferably linear polymers. Thepolymer may be a polycation, polyanion, or non-ionic polymer. Thebioactive agent, which may be a therapeutic agent, a diagnostic agent,or an adjuvant, preferably makes up at least 5%, 10%, 15%, 20%, 25%,30%, or even 35% by weight of the conjugate. In certain embodiments, therate of drug release is dependent primarily upon the rate of hydrolysis.In certain other embodiments, the rate of drug release is dependentprimarily on enzymatic cleavage.

The present invention provides cyclodextrin-containing polymericcompounds for use in drug delivery of these therapeutic agents. Theinvention also provides compounds for use in controlled drug deliverywhich are capable of releasing a therapeutic agent in a targeted,predictable, and controlled rate.

Accordingly, one aspect of the present invention is a polymer conjugatecomprising cyclodextrin moieties, a therapeutic agent, and an optionalligand targeting agent. The polymer may be linear or branched, and maybe formed via polycondensation of cyclodextrin-containing monomers,copolymerization between one or more cyclodextrin-containing monomersand one or more comonomers which do not contain cyclodextrin moieties.Furthermore, the present invention also contemplatescyclodextrin-containing polymers formed by grafting cyclodextrinmoieties to an already formed polymer. The cyclodextrin moietiescontemplated by the present invention include, but are not limited to,α, β, and γ cyclodextrins and oxidized forms thereof. Depending on thedrug/polymer ratio desired, the therapeutic agent may be attached to amonomer via an optional linker prior to the polymerization step, or maybe subsequently grafted onto the polymer via an optional linkerLikewise, the targeting ligand may be attached to a monomer via anoptional linker prior the polymerization step, or may be subsequentlygrafted onto the polymer via an optional linker, or may be attached tothe polymer as an inclusion complex or host-guest interactions.

To illustrate further, one embodiment of the invention is a polymericcompound represented by Formula I:

wherein

P represents a linear or branched polymer chain;

CD represents a cyclic moiety such as a cyclodextrin moiety;

L₁, L₂ and L₃, independently for each occurrence, may be absent orrepresent a linker group;

D, independently for each occurrence, represents a therapeutic agent ora prodrug thereof;

T, independently for each occurrence, represents a targeting ligand orprecursor thereof;

a, m, and v, independently for each occurrence, represent integers inthe range of 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

b represents an integer in the range of 1 to about 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5); and

n and w, independently for each occurrence, represents an integer in therange of 0 to about 30,000 (preferably <25,000, <20,000, <15,000,<10,000, <5,000, <1,000, <500, <100, <50, <25, <10, or even <5),

wherein either the polymer chain comprises cyclodextrin moieties or n isat least 1.

Another embodiment of the present invention is a compound represented byFormula II:

wherein

P represents a monomer unit of a polymer;

T, independently for each occurrence, represents a targeting ligand or aprecursor thereof;

L₆, L₇, L₈, L₉, and L₁₀, independently for each occurrence, may beabsent or represent a linker group;

CD, independently for each occurrence, represents a cyclic moiety suchas a cyclodextrin moiety or a derivative thereof;

D, independently for each occurrence, represents a therapeutic agent ora prodrug form thereof;

m, independently for each occurrence, represents an integer in the rangeof 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

o represents an integer in the range of 1 to about 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5); and

p, n, and q, independently for each occurrence, represent an integer inthe range of 0 to 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 toabout 2),

wherein CD and D are preferably each present at at least 1 location(preferably at least 5, 10, 25, 50 or even >100 locations) in thecompound.

Another embodiment of the present invention is a compound represented byFormula III:

wherein

CD represents a cyclic moiety such as a cyclodextrin moiety, orderivative thereof;

L₄, L₅, L₆, and L₇, independently for each occurrence, may be absent orrepresent a linker group;

D and D′, independently for each occurrence, represent the same ordifferent therapeutic agent or prodrugs thereof;

T and T′, independently for each occurrence, represent the same ordifferent targeting ligand or precursor thereof;

f and y, independently for each occurrence, represent an integer in therange of 1 and 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

g and z, independently for each occurrence, represent an integer in therange of 0 and 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 to about2); and

h represents an integer in the range of 1 and 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5),

wherein at least one occurrence (and preferably at least 5, 10, or evenat least 20, 50, or >100 occurrences) of g represents an integer greaterthan 0.

Another aspect of the present invention is a method for preparing thetherapeutic cyclodextrin-containing polymeric conjugates describedherein.

Another aspect of the present invention is a pharmaceutical compositioncomprising a compound or polymer as discussed above.

Another aspect of the present invention is a pharmaceutical dosage formcomprising a polymeric conjugate as described herein.

Another aspect of the present invention is a method for treating asubject comprising administering a therapeutically effective amount ofany of the polymeric conjugates described herein.

Another aspect of the present invention is a method of conducting apharmaceutical business comprising manufacturing, licensing, ordistributing kits containing or relating to any of the polymericconjugates described herein.

In certain embodiments, these therapeutic polymer conjugates improvedrug stability and/or solubility of the therapeutic agent when used invivo. Furthermore, by selecting from a variety of linker groups, thepolymer conjugates present methods for controlled release of thetherapeutic and/or bioactive agents, or improve the in vivo safetyand/or therapeutic efficacy of the therapeutic/bioactive agent. Incertain embodiments, the polymer conjugates are bioerodable orbiodegradable.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows strategies for varying polymer conjugates to tune theircharacteristics.

FIG. 2 demonstrates the effect of peptide tether length on drug releaserate for drug-loaded CD polymer.

FIG. 3 presents the effect that tethering camptothecin has on enhancingcamptothecin stability, e.g., inhibiting lactone ring-opening.

FIG. 4 shows lactone ring opening studies in pH 7.4 KH2PO4 buffer.

FIGS. 5 a and 5 b show polymerization control by adjustingpolymerization time.

FIG. 6 illustrates CPT release from HG6 and HGGG6 at 37° C. after 24 hin buffer solutions with pHs ranging from 1.1 to 13.1.

FIG. 7 Displays HPLC analysis of degradation of CD-BisCys-SS-Peg3400Polymer

FIG. 8 Shows the tumor growth curve as a function of time for the D5W,CPT, irinotecan, LGGG10 at its highest non-toxic dose tested (18 mgCPT/kg), and the other three conjugates with high MW polymer (HGGG6,HG6, HGGG10) at their MTDs in xenograft mice.

FIG. 9 presents the median tumor growth curves for HGGG6, HG6 and HGGG10in xenograft mice.

FIG. 10 presents the medium tumor growth curves for LGGG10 and HGGG10each dosed at 9 mg CPT/kg in xenograft mice.

FIG. 11 presents the mean body weight (MBW) losses as a function of timeplotted for D5W, CPT, irinotecan and the three conjugates containinghigh MW polymer at their MTDs in xenograft mice.

FIG. 12 shows the correlation of CPT concentration (ng/mg tissue) totumor size (in mg) in xenograft mice.

FIG. 13 is a schematic depicting a general strategy for synthesizinglinear, branched or grafter cyclodextrin-containing polymers (CDPs) forloading a therapeutic agent, and an optional targeting ligand.

FIG. 14 is a schematic depicting a general scheme for graft polymers.

FIG. 15 is a schematic depicting a general scheme of preparing linearCDPs.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The present invention relates to novel compositions of therapeuticcyclodextrin-containing polymeric compounds designed for drug deliveryof therapeutic agents. In certain embodiments, thesecyclodextrin-containing polymers improve drug stability and/orsolubility, and/or reduce toxicity, and/or improve efficacy of the smallmolecule therapeutic when used in vivo. In certain embodiments, thepolymers can be used for delivery of therapeutics such as camptothecin,taxol, doxorubicin, and amphotericin. Furthermore, by selecting from avariety of linker groups, and/or targeting ligands, the rate of drugrelease from the polymers can be attenuated for controlled delivery. Theinvention also relates to methods of treating subjects with thetherapeutic compositions described herein. The invention further relatesto methods for conducting a pharmaceutical business comprisingmanufacturing, licensing, or distributing kits containing or relating tothe polymeric compounds described herein.

More generally, the present invention provides water-soluble,biocompatible polymer conjugates comprising a water-soluble,biocompatible polymer covalently attached to bioactive moieties throughattachments that are cleaved under biological conditions to release thebioactive moieties. In certain such embodiments, the polymer comprisescyclic moieties alternating with linker moieties that connect the cyclicstructures, e.g., into linear or branched polymers, preferably linearpolymers. The cyclic moieties may be any suitable cyclic structures,such as cyclodextrins, crown ethers (e.g., 18-crown-6, 15-crown-5,12-crown-4, etc.), cyclic oligopeptides (e.g., comprising from 5 to 10amino acid residues), cryptands or cryptates (e.g., cryptand [2.2.2],cryptand-2,1,1, and complexes thereof), calixarenes, or cavitands, orany combination thereof. Preferably, the cyclic structure is (or ismodified to be) water-soluble. In certain embodiments, e.g., where alinear polymer is desired, the cyclic structure is selected such thatunder polymerization conditions, exactly two moieties of each cyclicstructure are reactive with the linker moieties, such that the resultingpolymer comprises (or consists essentially of) an alternating series ofcyclic moieties and linker moieties, such as at least four of each typeof moiety. Suitable difunctionalized cyclic moieties include many thatare commercially available and/or amenable to preparation usingpublished protocols. In certain embodiments, conjugates are soluble inwater to a concentration of at least 0.1 g/mL, preferably at least 0.25g/mL.

The polymer may be a polycation, polyanion, or non-ionic polymer. Apolycationic or polyanionic polymer has at least one site that bears apositive or negative charge, respectively. In certain such embodiments,at least one of the linker moiety and the cyclic moiety comprises such acharged site, so that every occurrence of that moiety includes a chargedsite.

The bioactive agent, which may be a therapeutic agent, a diagnosticagent, or an adjuvant (such as a radiosensitizer, or a compound thatlacks significant activity administered alone but that potentiates theactivity of another therapeutic agent), preferably makes up at least 5%,10%, 15%, 20%, 25%, 30%, or even 35% by weight of the conjugate. Inpreferred embodiments, administration of the polymer to a patientresults in release of the bioactive agent over a period of at least 6hours, preferably at least 12 or 18 hours. For example, the agent may bereleased over a period of time ranging from 6 hours to a month, 6 hoursto two weeks, 6 hours to 3 days, etc. In certain embodiments, the rateof drug release is dependent primarily upon the rate of hydrolysis (asopposed to enzymatic cleavage), e.g., the rate of release changes byless than a factor of 5, preferably less than a factor of 2, in thepresence of hydrolytic enzymes. In other embodiments, the rate of drugrelease may be dependent primarily on the rate of enzymatic cleavage.

Polymeric conjugates of the present invention may be useful to improvesolubility and/or stability of a bioactive/therapeutic agent, reducedrug-drug interactions, reduce interactions with blood elementsincluding plasma proteins, reduce or eliminate immunogenicity, protectthe agent from metabolism, modulate drug-release kinetics, improvecirculation time, improve drug half-life (e.g., in the serum, or inselected tissues, such as tumors), attenuate toxicity, improve efficacy,normalize drug metabolism across subjects of different species,ethnicities, and/or races, and/or provide for targeted delivery intospecific cells or tissues. Poorly soluble and/or toxic compounds maybenefit particularly from incorporation into polymeric compounds of theinvention.

II. Definitions

(a) General Terms

An ‘adjuvant’, as the term is used herein, is a compound that has littleor no therapeutic value on its own, but increases the effectiveness of atherapeutic agent. Exemplary adjuvants include radiosensitizers,transfection-enhancing agents (such as chloroquine and analogs thereof),chemotactic agents and chemoattractants, peptides that modulate celladhesion and/or cell mobility, cell permeabilizing agents, inhibitors ofmultidrug resistance and/or efflux pumps, etc.

The term “agonist”, as used herein, is meant to refer to an agent thatmimics or up-regulates (e.g., potentiates or supplements) thebioactivity of a protein of interest, or an agent that facilitates orpromotes (e.g., potentiates or supplements) an interaction amongpolypeptides or between a polypeptide and another molecule (e.g., asteroid, hormone, nucleic acids, small molecules etc.). An agonist canbe a wild-type protein or derivative thereof having at least onebioactivity of the wild-type protein. An agonist can also be a smallmolecule that up-regulates the expression of a gene or which increasesat least one bioactivity of a protein. An agonist can also be a proteinor small molecule which increases the interaction of a polypeptide ofinterest with another molecule, e.g., a target peptide or nucleic acid.

“Antagonist” as used herein is meant to refer to an agent thatdown-regulates (e.g., suppresses or inhibits) the bioactivity of aprotein of interest, or an agent that inhibits/suppresses or reduces(e.g., destabilizes or decreases) interaction among polypeptides orother molecules (e.g., steroids, hormones, nucleic acids, etc.). Anantagonist can also be a compound that down-regulates the expression ofa gene of interest or which reduces the amount of the wild-type proteinpresent. An antagonist can also be a protein or small molecule whichdecreases or inhibits the interaction of a polypeptide of interest withanother molecule, e.g., a target peptide or nucleic acid.

The terms “biocompatible polymer” and “biocompatibility” when used inrelation to polymers are art-recognized. For example, biocompatiblepolymers include polymers that are neither themselves toxic to the host(e.g., an animal or human), nor degrade (if the polymer degrades) at arate that produces monomeric or oligomeric subunits or other byproductsat toxic concentrations in the host. In certain embodiments of thepresent invention, biodegradation generally involves degradation of thepolymer in an organism, e.g., into its monomeric subunits, which may beknown to be effectively non-toxic. Intermediate oligomeric productsresulting from such degradation may have different toxicologicalproperties, however, or biodegradation may involve oxidation or otherbiochemical reactions that generate molecules other than monomericsubunits of the polymer. Consequently, in certain embodiments,toxicology of a biodegradable polymer intended for in vivo use, such asimplantation or injection into a patient, may be determined after one ormore toxicity analyses. It is not necessary that any subject compositionhave a purity of 100% to be deemed biocompatible. Hence, a subjectcomposition may comprise 99%, 98%, 97%, 96%, 95%, 90% 85%, 80%, 75% oreven less of biocompatible polymers, e.g., including polymers and othermaterials and excipients described herein, and still be biocompatible.

To determine whether a polymer or other material is biocompatible, itmay be necessary to conduct a toxicity analysis. Such assays are wellknown in the art. One example of such an assay may be performed withlive carcinoma cells, such as GT3TKB tumor cells, in the followingmanner: the sample is degraded in 1 M NaOH at 37° C. until completedegradation is observed. The solution is then neutralized with 1 M HCl.About 200 μL of various concentrations of the degraded sample productsare placed in 96 -well tissue culture plates and seeded with humangastric carcinoma cells (GT3TKB) at 104/well density. The degradedsample products are incubated with the GT3TKB cells for 48 hours. Theresults of the assay may be plotted as % relative growth vs.concentration of degraded sample in the tissue-culture well. Inaddition, polymers and formulations of the present invention may also beevaluated by well-known in vivo tests, such as subcutaneousimplantations in rats to confirm that they do not cause significantlevels of irritation or inflammation at the subcutaneous implantationsites.

The term “biodegradable” is art-recognized, and includes polymers,compositions and formulations, such as those described herein, that areintended to degrade during use. Biodegradable polymers typically differfrom non-biodegradable polymers in that the former may be degradedduring use. In certain embodiments, such use involves in vivo use, suchas in vivo therapy, and in other certain embodiments, such use involvesin vitro use. In general, degradation attributable to biodegradabilityinvolves the degradation of a biodegradable polymer into its componentsubunits, or digestion, e.g., by a biochemical process, of the polymerinto smaller, non-polymeric subunits. In certain embodiments, twodifferent types of biodegradation may generally be identified. Forexample, one type of biodegradation may involve cleavage of bonds(whether covalent or otherwise) in the polymer backbone. In suchbiodegradation, monomers and oligomers typically result, and even moretypically, such biodegradation occurs by cleavage of a bond connectingone or more of subunits of a polymer. In contrast, another type ofbiodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to sidechain or that connects a side chain to thepolymer backbone. For example, a therapeutic agent or other chemicalmoiety attached as a side chain to the polymer backbone may be releasedby biodegradation. In certain embodiments, one or the other or bothgeneral types of biodegradation may occur during use of a polymer.

As used herein, the term “biodegradation” encompasses both general typesof biodegradation. The degradation rate of a biodegradable polymer oftendepends in part on a variety of factors, including the chemical identityof the linkage responsible for any degradation, the molecular weight,crystallinity, biostability, and degree of cross-linking of suchpolymer, the physical characteristics (e.g., shape and size) of animplant, and the mode and location of administration. For example, thegreater the molecular weight, the higher the degree of crystallinity,and/or the greater the bio stability, the biodegradation of anybiodegradable polymer is usually slower. The term “biodegradable” isintended to cover materials and processes also termed “bioerodible”.

In certain embodiments wherein the biodegradable polymer also has atherapeutic agent or other material associated with it, thebiodegradation rate of such polymer may be characterized by a releaserate of such materials. In such circumstances, the biodegradation ratemay depend on not only the chemical identity and physicalcharacteristics of the polymer, but also on the identity of material(s)incorporated therein. Degradation of the subject compositions includesnot only the cleavage of intramolecular bonds, e.g., by oxidation and/orhydrolysis, but also the disruption of intermolecular bonds, such asdissociation of host/guest complexes by competitive complex formationwith foreign inclusion hosts.

In certain embodiments, polymeric formulations of the present inventionbiodegrade within a period that is acceptable in the desiredapplication. In certain embodiments, such as in vivo therapy, suchdegradation occurs in a period usually less than about five years, oneyear, six months, three months, one month, fifteen days, five days,three days, or even one day on exposure to a physiological solution witha pH between 6 and 8 having a temperature of between 25 and 37° C. Inother embodiments, the polymer degrades in a period of between about onehour and several weeks, depending on the desired application.

As used herein the term “bioerodable” refers to polymers which deliversustained effective amounts of therapeutic agent to target tissue overdesired extended periods of time. Thus, a polymer according to theinvention in the biological environment of host tissue and the like, inone aspect, is subjected to hydrolytic enzymes and oxidative speciesunder, and in proportion to, the host's inflammatory response. Thisresults in release of the therapeutic agent via the breaking of thecovalent linked bonds. Thus, in certain embodiments, the materials ofthe invention utilize the mammal's own wound-healing repair process inbeing degraded thereby, as hereinbefore described.

The biodegradable polymers polylactic acid, polyglycolic acid, andpolylactic-glycolic acid copolymer (PLGA), have been investigatedextensively for nanoparticle formulation. These polymers are polyestersthat, upon implantation in the body, undergo simple hydrolysis. Theproducts of such hydrolysis are biologically compatible andmetabolizable moieties (e.g., lactic acid and glycolic acid), which areeventually removed from the body by the citric acid cycle. Polymerbiodegradation products are formed at a very slow rate, and hence do notaffect normal cell function. Several implant studies with these polymershave proven safe in drug delivery applications, used in the form ofmatrices, microspheres, bone implant materials, surgical sutures, andalso in contraceptive applications for long-term effects. These polymersare also used as graft materials for artificial organs, and recently asbasement membranes in tissue engineering investigations. Nature Med.824-826 (1996). Thus, these polymers have been time-tested in variousapplications and proven safe for human use. Most importantly, thesepolymers are FDA-approved for human use.

When polymers are used for delivery of pharmacologically active agentsin vivo, it is essential that the polymers themselves be nontoxic andthat they degrade into non-toxic degradation products as the polymer iseroded by the body fluids. Many synthetic biodegradable polymers,however, yield oligomers and monomers upon erosion in vivo thatadversely interact with the surrounding tissue. D. F. Williams, J.Mater. Sci. 1233 (1982). To minimize the toxicity of the intact polymercarrier and its degradation products, polymers have been designed basedon naturally occurring metabolites. Probably the most extensivelystudied examples of such polymers are the polyesters derived from lacticor glycolic acid and polyamides derived from amino acids.

A number of bioerodable or biodegradable polymers are known and used forcontrolled release of pharmaceuticals. Such polymers are described in,for example, U.S. Pat. Nos. 4,291,013; 4,347,234; 4,525,495; 4,570,629;4,572,832; 4,587,268; 4,638,045; 4,675,381; 4,745,160; and 5,219,980.

A biohydrolyzable bond (e.g., ester, amide, carbonate, carbamates, orimide) refers to a bond that is cleaved (e.g., an ester is cleaved toform a hydroxyl and a carboxylic acid) under physiological conditions.Physiological conditions include the acidic and basic environments ofthe digestive tract (e.g., stomach, intestines, etc.), acidicenvironment of a tumor, enzymatic cleavage, metabolism, and otherbiological processes, and preferably refer to physiological conditionsin a vertebrate, such as a mammal.

As used herein the terms “comonomer A precursor”, “linker”, “linkergroup”, and “linker moiety” refer to any straight chain or branched,symmetric or asymmetric compound which upon reaction with a cyclodextrinmonomer precursor or other suitable cyclic moiety links two suchmoieties together. In certain embodiments, a comonomer A precursor is acompound containing at least two functional groups through whichreaction and thus linkage of the cyclodextrin monomers can be achieved.Examples of functional groups, which may be the same or different,terminal or internal, of each comonomer A precursor include, but are notlimited, to amino, acid, imidazole, hydroxyl, thio, acyl halide, —C═C—,or —C≡C— groups and derivatives thereof. In preferred embodiments, thetwo functional groups are the same and are located at termini of thecomonomer. In certain embodiments, a comonomer A precursor contains oneor more pendant groups with at least one functional group through whichreaction and thus linkage of therapeutic agent or targeting ligand canbe achieved, or branched polymerization can be achieved. Examples offunctional groups, which may be the same or different, terminal orinternal, of each comonomer A precursor pendant group include, but arenot limited, to amino, acid, imidazole, hydroxyl, thiol, acyl halide,ethylene, and ethyne groups and derivatives thereof. In certainembodiments, the pendant group is a (un)substituted branched, cyclic orstraight chain C1-C10 (preferably C1-C6) alkyl, or arylalkyl optionallycontaining one or more heteroatoms, e.g., N, O, S, within the chain orring.

Upon copolymerization of a comonomer A precursor with a cyclodextrinmonomer precursor, two cyclodextrin monomers may be linked together byjoining the primary hydroxyl side of one cyclodextrin monomer with theprimary hydroxyl side of another cyclodextrin monomer, by joining thesecondary hydroxyl side of one cyclodextrin monomer with the secondaryhydroxyl side of another cyclodextrin monomer, or by joining the primaryhydroxyl side of one cyclodextrin monomer with the secondary hydroxylside of another cyclodextrin monomer. Accordingly, combinations of suchlinkages may exist in the final copolymer. Both the comonomer Aprecursor and the comonomer A of the final copolymer may be neutral,cationic (e.g., by containing protonated groups such as, for example,quaternary ammonium groups), or anionic (e.g., by containingdeprotonated groups, such as, for example, sulfate, phosphate, borinateor carboxylate). The charge of comonomer A of the copolymer may beadjusted by adjusting pH conditions. Examples of suitable comonomer Aprecursors include, but are not limited to succinimide (e.g.,dithiobis(succinimidyl propionate) DSP, and dissucinimidyl suberate(DSS)), glutamates, and aspartates).

The cyclodextrin-containing polymers of the present invention may belinear, branched or grafted. As used herein, the term “linearcyclodextrin-containing polymer” refers to a polymer comprising (α, β,or γ) cyclodextrin molecules, or derivatives thereof which are insertedwithin a polymer chain. As used herein, the term “graftedcyclodextrin-containing polymer” refers to a polymer comprising (α, β,or γ) cyclodextrin molecules, or derivatives thereof which are pendantoff of the polymer chain. The term “graft polymer” as used herein refersto a polymer molecule which has additional moieties attached as pendentgroups along a polymer backbone. The term “graft polymerization” denotesa polymerization in which a side chain is grafted onto a polymer chain,which side chain consists of one or several other monomers. Theproperties of the graft copolymer obtained such as, for example,solubility, melting point, water absorption, wettability, mechanicalproperties, adsorption behavior, etc., deviate more or less sharply fromthose of the initial polymer as a function of the type and amount of thegrafted monomers. The term “grafting ratio”, as used herein, means theweight percent of the amount of the monomers grafted based on the weightof the polymer. As used herein, a branched cyclodextrin-containingpolymer refers to a polymer backbone with a plurality of branch points,wherein each branch point is a starting point of yet another strand ofthe polymer backbone, and each section of polymer backbone may have aplurality of (α, β, or γ) cyclodextrin molecules, or derivativesthereof, inserted into or grafted onto the chain.

The term “cyclodextrin moiety” refers to (α, β, or γ) cyclodextrinmolecules or derivatives thereof, which may be in their oxidized orreduced forms. Cyclodextrin moieties may comprise optional linkers.Optional therapeutic agents and/or targeting ligands may be furtherlinked to these moieties via an optional linker. The linkage may becovalent (optionally via biohydrolyzable bonds, e.g., esters, amides,carbamates, and carbonates) or may be a host-guest complex between thecyclodextrin derivative and the therapeutic agent and/or targetingligand or the optional linkers of each. Cyclodextrin moieties mayfurther include one or more carbohydrate moieties, preferably simplecarbohydrate moieties such as galactose, attached to the cyclic core,either directly (i.e., via a carbohydrate linkage) or through a linkergroup.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

An ‘effective amount’ of a subject compound, with respect to the subjectmethod of treatment, refers to an amount of the therapeutic in apreparation which, when applied as part of a desired dosage regimenprovides a benefit according to clinically acceptable standards for thetreatment or prophylaxis of a particular disorder.

The term “healthcare providers” refers to individuals or organizationsthat provide healthcare services to a person, community, etc. Examplesof “healthcare providers” include doctors, hospitals, continuing careretirement communities, skilled nursing facilities, subacute carefacilities, clinics, multispecialty clinics, freestanding ambulatorycenters, home health agencies, and HMO's.

“Instruction(s)” as used herein means documents describing relevantmaterials or methodologies pertaining to a kit. These materials mayinclude any combination of the following: background information, listof components and their availability information (purchase information,etc.), brief or detailed protocols for using the kit, trouble-shooting,references, technical support, and any other related documents.Instructions can be supplied with the kit or as a separate membercomponent, either as a paper form or an electronic form which may besupplied on computer readable memory device or downloaded from aninternet website, or as recorded presentation. Instructions can compriseone or multiple documents, and are meant to include future updates.

“Kit” as used herein means a collection of at least two componentsconstituting the kit. Together, the components constitute a functionalunit for a given purpose. Individual member components may be physicallypackaged together or separately. For example, a kit comprising aninstruction for using the kit may or may not physically include theinstruction with other individual member components. Instead, theinstruction can be supplied as a separate member component, either in apaper form or an electronic form which may be supplied on computerreadable memory device or downloaded from an internet website, or asrecorded presentation.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human subject.

The “polymerizations” of the present invention include radical, anionic,and cationic mechanisms, as well as reactions of bifunctional molecules(analogous to the formation of nylon, e.g., reacting molecules each ofwhich bears two or more different reactive moieties that react with eachother (but, preferably, are disfavored from reacting intramolecularly bysteric, conformational, or other constraints), or reacting two or moredifferent compounds, each compound bearing two or more reactive moietiesthat react only with reactive moieties of different compounds (i.e.,intermolecularly)), as well as metal-catalyzed polymerizations such asolefin metathesis, and other polymerization reactions known to those ofskill in the art.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the frequency of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

As used herein, the terms “therapeutic agent” include any synthetic ornaturally occurring biologically active compound or composition ofmatter which, when administered to an organism (human or nonhumananimal), induces a desired pharmacologic, immunogenic, and/orphysiologic effect by local and/or systemic action. The term thereforeencompasses those compounds or chemicals traditionally regarded asdrugs, vaccines, and biopharmaceuticals including molecules such asproteins, peptides, hormones, nucleic acids, gene constructs and thelike. More particularly, the term “therapeutic agent” includes compoundsor compositions for use in all of the major therapeutic areas including,but not limited to, adjuvants; anti-infectives such as antibiotics andantiviral agents; analgesics and analgesic combinations, anorexics,anti-inflammatory agents, anti-epileptics, local and generalanesthetics, hypnotics, sedatives, antipsychotic agents, neurolepticagents, antidepressants, anxiolytics, antagonists, neuron blockingagents, anticholinergic and cholinomimetic agents, antimuscarinic andmuscarinic agents, antiadrenergics, antiarrhythmics, antihypertensiveagents, hormones, and nutrients, antiarthritics, antiasthmatic agents,anticonvulsants, antihistamines, antinauseants, antineoplastics,antipruritics, antipyretics; antispasmodics, cardiovascular preparations(including calcium channel blockers, beta-blockers, beta-agonists andantiarrythmics), antihypertensives, diuretics, vasodilators; centralnervous system stimulants; cough and cold preparations; decongestants;diagnostics; hormones; bone growth stimulants and bone resorptioninhibitors; immunosuppressives; muscle relaxants; psychostimulants;sedatives; tranquilizers; proteins, peptides, and fragments thereof(whether naturally occurring, chemically synthesized or recombinantlyproduced); and nucleic acid molecules (polymeric forms of two or morenucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA)including both double- and single-stranded molecules, gene constructs,expression vectors, antisense molecules and the like), small molecules(e.g., doxorubicin) and other biologically active macromolecules suchas, for example, proteins and enzymes. The agent may be a biologicallyactive agent used in medical, including veterinary, applications and inagriculture, such as with plants, as well as other areas. The termtherapeutic agent also includes without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness; orsubstances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

As used herein the term “low aqueous solubility” refers to waterinsoluble compounds having poor solubility in water, that is <5 mg/ml atphysiological pH (6.5-7.4). Preferably, their water solubility is <1mg/ml, more preferably <0.1 mg/ml. It is desirable that the drug isstable in water as a dispersion; otherwise a lyophilized or spray-driedsolid form may be desirable.

Examples of some preferred water-insoluble drugs includeimmunosuppressive agents such as cyclosporins including cyclosporine(cyclosporin A), immunoactive agents, antiviral and antifungal agents,antineoplastic agents, analgesic and anti-inflammatory agents,antibiotics, anti-epileptics, anesthetics, hypnotics, sedatives,antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics,anticonvulsant agents, antagonists, neuron blocking agents,anticholinergic and cholinomimetic agents, antimuscarinic and muscarinicagents, antiadrenergic and antiarrhythmics, antihypertensive agents,hormones, and nutrients. A detailed description of these and othersuitable drugs may be found in Remington's Pharmaceutical Sciences, 18thedition, 1990, Mack Publishing Co. Philadelphia, Pa.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “therapeutically effective amount” of a compound, with respect to amethod of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

A “therapeutically effective daily dosage” of a compound, with respectto a method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired daily dosageregimen (to a mammal, preferably a human) alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

(b) Chemical Terms

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain radicals. As used herein, the term “aliphatic group” refersto a straight chain, branched-chain, or cyclic aliphatic hydrocarbongroup and includes saturated and unsaturated aliphatic groups, such asan alkyl group, an alkenyl group, and an alkynyl group.

Alkyl refers to a fully saturated branched or unbranched carbon chainradical having the number of carbon atoms specified, or up to 30 carbonatoms if no specification is made. For example, alkyl of 1 to 8 carbonatoms refers to radicals such as methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, and octyl, and those radicals which are positionalisomers of these radicals. Alkyl of 10 to 30 carbon atoms includesdecyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyland tetracosyl. In preferred embodiments, a straight chain or branchedchain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30for straight chains, C3-C30 for branched chains), and more preferably 20or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, a cyano, a nitro, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude substituted and unsubstituted forms of amino, azido, imino,amido, phosphoryl (including phosphonate and phosphinate), sulfonyl(including sulfate, sulfonamido, sulfamoyl and sulfonate), and silylgroups, as well as ethers, alkylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF3, —CN and the like. Exemplarysubstituted alkyls are described below. Cycloalkyls can be furthersubstituted with alkyls, alkenyls, alkoxyls, alkylthios, aminoalkyls,carbonyl-substituted alkyls, —CF3, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl”, asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application,preferred alkyl groups are lower alkyls. In preferred embodiments, asubstituent designated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH2)m-R1, wherein m and R1 are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

Alkenyl refers to any branched or unbranched unsaturated carbon chainradical having the number of carbon atoms specified, or up to 26 carbonatoms if no limitation on the number of carbon atoms is specified; andhaving 1 or more double bonds in the radical. Alkenyl of 6 to 26 carbonatoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,where the unsaturated bond(s) can be located anywhere in the radical andcan have either the (Z) or the (E) configuration about the doublebond(s).

Alkynyl refers to hydrocarbyl radicals of the scope of alkenyl, buthaving 1 or more triple bonds in the radical.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH2)m-R1,where m and R1 are described below.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formulae:

wherein R3, R5 and R6 each independently represent a hydrogen, an alkyl,an alkenyl, —(CH2)m-R1, or R3 and R5 taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure; R1 represents an alkenyl, aryl, cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In preferred embodiments, only one of R3or R5 can be a carbonyl, e.g., R3, R5 and the nitrogen together do notform an imide. In even more preferred embodiments, R3 and R5 (andoptionally R6) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH2)m-R1. Thus, the term “alkylamine” as used herein meansan amine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R3 and R5 is an alkylgroup. In certain embodiments, an amino group or an alkylamine is basic,meaning it has a pKa>7.00. The protonated forms of these functionalgroups have pKas relative to water above 7.00.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R7represents a hydrogen, an alkyl, an alkenyl, —(CH2)m-R1 or apharmaceutically acceptable salt, R8 represents a hydrogen, an alkyl, analkenyl or —(CH2)m-R1, where m and R1 are as defined above. Where X isan oxygen and R7 or R8 is not hydrogen, the formula represents an“ester”. Where X is an oxygen, and R7 is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R7 is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R8 is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R7 or R8 is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R7 is hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R8 ishydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R7 is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R7 is hydrogen, theabove formula represents an “aldehyde” group.

The term “derivatized” refers to chemically modifying molecules. Thechemical modifications may be artificial such as formation of drugs,natural such as formation of metabolites. The skilled artisan wouldreadily recognize the variety of ways molecules may be modified, such asoxidations, reductions, electrophilic/nucleophilic substitutions,alkylations, ester/amide formations and the like. For example,cyclodextrins of the present invention may be chemically modified byamination, tosylation, or iodination prior to covalently attaching themto the polymeric matrix Likewise, therapeutic agents may be chemicallymodified by preparing prodrugs (e.g., glycine-camptothecin).

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN,or the like.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The term “hydrocarbyl” refers to a monovalent hydrocarbon radicalcomprised of carbon chains or rings of up to 26 carbon atoms to whichhydrogen atoms are attached. The term includes alkyl, cycloalkyl,alkenyl, alkynyl and aryl groups, groups which have a mixture ofsaturated and unsaturated bonds, carbocyclic rings and includescombinations of such groups. It may refer to straight chain,branched-chain, cyclic structures or combinations thereof.

The term “hydrocarbylene” refers to a divalent hydrocarbyl radical.Representative examples include alkylene, phenylene, or cyclohexylene.Preferably, the hydrocarbylene chain is fully saturated and/or has achain of 1-10 carbon atoms.

As used herein, the term “nitro” means —NO2; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO2-.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Ms represent methyl, ethyl, phenyl, andmethanesulfonyl, respectively. A more comprehensive list of theabbreviations utilized by organic chemists of ordinary skill in the artappears in the first issue of each volume of the Journal of OrganicChemistry; this list is typically presented in a table entitled StandardList of Abbreviations. The abbreviations contained in said list, and allabbreviations utilized by organic chemists of ordinary skill in the artare hereby incorporated by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, (R)- and(S)-enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivatization with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts may be formedwith an appropriate optically active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Applications of Method and Compositions

(a) Exemplary Compositions

The present invention includes polymer conjugates, such ascyclodextrin-containing polymer conjugates, wherein one or moretherapeutic/bioactive agents are covalently attached. In certainembodiments, the therapeutic agent is a small molecule, a macromolecule,an antibody, a peptide, a protein, an enzyme, a nucleic acid, or apolymer that has therapeutic function. The polymers include linear orbranched cyclodextrin-containing polymers and polymers grafted withcyclodextrin. Exemplary cyclodextrin-containing polymers that may bemodified as described herein are taught in U.S. Pat. No. 6,509,323,published U.S. application No. 20020151523, and U.S. patent applicationserial Nos. 60/417,373, and 10/372,723. These polymers are useful ascarriers for small molecule therapeutic delivery, and may improve drugstability and solubility when used in vivo.

Accordingly, one embodiment of present invention is a polymeric compoundrepresented by Formula I:

wherein

P represents a linear or branched polymer chain;

CD represents a cyclic moiety such as a cyclodextrin moiety;

L₁, L₂ and L₃, independently for each occurrence, may be absent orrepresent a linker group;

D, independently for each occurrence, represents a therapeutic agent ora prodrug thereof;

T, independently for each occurrence, represents a targeting ligand orprecursor thereof;

a, m, and v, independently for each occurrence, represent integers inthe range of 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

n and w, independently for each occurrence, represent an integer in therange of 0 to about 30,000 (preferably <25,000, <20,000, <15,000,<10,000, <5,000, <1,000, <500, <100, <50, <25, <10, or even <5); and

b represents an integer in the range of 1 to about 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5),

wherein either P comprises cyclodextrin moieties or n is at least 1.

In certain embodiments, P contains a plurality of cyclodextrin moietieswithin the polymer chain as opposed to the cyclodextrin moieties beinggrafted on to pendant groups off of the polymeric chain. Thus in certainembodiments, the polymer chain of formula I further comprises n′ unitsof U, wherein n′ represents an integer in the range of 1 to about 30,000(preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500,<100, <50, <25, <10, or even <5); and U is represented by the generalformula:

wherein

CD represents a cyclic moiety, such as a cyclodextrin moiety, orderivative thereof;

L₄, L₅, L₆, and L₇, independently for each occurrence, may be absent orrepresent a linker group;

D and D′, independently for each occurrence, represent the same ordifferent therapeutic agent or prodrug forms thereof;

T and T′, independently for each occurrence, represent the same ordifferent targeting ligand or precursor thereof;

f and y, independently for each occurrence, represent an integer in therange of 1 and 10; and

g and z, independently for each occurrence, represent an integer in therange of 0 and 10.

In preferred embodiments, L₄ and L₇ represent linker groups.

In certain embodiments, the polymer may be selected frompolysaccharides, and other non-protein biocompatible polymers, andcombinations thereof, that contain at least one terminal hydroxyl group,such as polyvinylpyrrollidone, poly(oxyethylene)glycol (PEG),polysuccinic anhydride, polysebacic acid, PEG-phosphate, polyglutamate,polyethylenimine, maleic anhydride divinylether (DIVMA), cellulose,pullulans, inulin, polyvinyl alcohol (PVA),N-(2-hydroxypropyl)methacrylamide (HPMA), dextran and hydroxyethylstarch (HES), and have optional pendant groups for grafting therapeuticagents, targeting ligands and/or cyclodextrin moieties. In certainembodiments, the polymer may be biodegradable such as poly(lactic acid),poly(glycolic acid), poly(alkyl 2-cyanoacrylates), polyanhydrides, andpolyorthoesters, or bioerodible such as polylactide-glycolidecopolymers, and derivatives thereof, non-peptide polyaminoacids,polyiminocarbonates, poly alpha-amino acids, polyalkyl-cyano-acrylate,polyphosphazenes or acyloxymethyl poly aspartate and polyglutamatecopolymers and mixtures thereof.

Another embodiment of the invention is a polymeric compound representedby Formula II:

wherein

P represents a monomer unit of a polymer;

T, independently for each occurrence, represents a targeting ligand or aprecursor thereof;

L₆, L₇, L₈, L₉, and L₁₀, independently for each occurrence, may beabsent or represent a linker group;

CD, independently for each occurrence, represents a cyclodextrin moietyor a derivative thereof;

D, independently for each occurrence, represents a therapeutic agent ora prodrug form thereof;

m, independently for each occurrence, represents an integer in the rangeof 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

o represents an integer in the range of 1 to about 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5); and

p, n, and q, independently for each occurrence, represent an integer inthe range of 0 to 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 toabout 2),

wherein CD and D are preferably each present at at least 1 location(preferably at least 5, 10, 25, or even 50 or 100 locations) in thecompound.

Another embodiment of the invention is a compound represented by FormulaIII:

wherein

CD represents a cyclic moiety, such as a cyclodextrin moiety, orderivative thereof;

L₄, L₅, L₆, and L₇, independently for each occurrence, may be absent orrepresent a linker group;

D and D′, independently for each occurrence, represent the same ordifferent therapeutic agent or prodrugs thereof;

T and T′, independently for each occurrence, represent the same ordifferent targeting ligand or precursor thereof;

f and y, independently for each occurrence, represent an integer in therange of 1 and 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

g and z, independently for each occurrence, represent an integer in therange of 0 and 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 to about2); and

h represents an integer in the range of 1 and 30,000 (preferably<25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50,<25, <10, or even <5),

wherein at least one occurrence (and preferably at least 5, 10, or evenat least 20, 50, or 100 occurrences) of g represents an integer greaterthan 0.

In preferred embodiments, L4 and L7 represent linker groups.

In certain embodiments, the underlying polymers are linearcyclodextrin-containing polymers, e.g., the polymer backbone includescyclodextrin moieties. For example, the polymer may be a water-soluble,linear cyclodextrin polymer produced by providing at least onecyclodextrin derivative modified to bear one reactive site at each ofexactly two positions, and reacting the cyclodextrin derivative with alinker having exactly two reactive moieties capable of forming acovalent bond with the reactive sites under polymerization conditionsthat promote reaction of the reactive sites with the reactive moietiesto form covalent bonds between the linker and the cyclodextrinderivative, whereby a linear polymer comprising alternating units ofcyclodextrin derivatives and linkers is produced. Alternatively thepolymer may be a water-soluble, linear cyclodextrin polymer having alinear polymer backbone, which polymer comprises a plurality ofsubstituted or unsubstituted cyclodextrin moieties and linker moietiesin the linear polymer backbone, wherein each of the cyclodextrinmoieties, other than a cyclodextrin moiety at the terminus of a polymerchain, is attached to two of said linker moieties, each linker moietycovalently linking two cyclodextrin moieties. In yet another embodiment,the polymer is a water-soluble, linear cyclodextrin polymer comprising aplurality of cyclodextrin moieties covalently linked together by aplurality of linker moieties, wherein each cyclodextrin moiety, otherthan a cyclodextrin moiety at the terminus of a polymer chain, isattached to two linker moieties to form a linear cyclodextrin polymer.

The linker group(s) may be an alkylene chain, a polyethylene glycol(PEG) chain, polysuccinic anhydride, poly-L-glutamic acid,poly(ethyleneimine), an oligosaccharide, an amino acid chain, or anyother suitable linkage. In certain embodiments, the linker group itselfcan be stable under physiological conditions, such as an alkylene chain,or it can be cleavable under physiological conditions, such as by anenzyme (e.g., the linkage contains a peptide sequence that is asubstrate for a peptidase), or by hydrolysis (e.g., the linkage containsa hydrolyzable group, such as an ester or thioester). The linker groupscan be biologically inactive, such as a PEG, polyglycolic acid, orpolylactic acid chain, or can be biologically active, such as an oligo-or polypeptide that, when cleaved from the moieties, binds a receptor,deactivates an enzyme, etc. Various oligomeric linker groups that arebiologically compatible and/or bioerodible are known in the art, and theselection of the linkage may influence the ultimate properties of thematerial, such as whether it is durable when implanted, whether itgradually deforms or shrinks after implantation, or whether it graduallydegrades and is absorbed by the body. The linker group may be attachedto the moieties by any suitable bond or functional group, includingcarbon-carbon bonds, esters, ethers, amides, amines, carbonates,carbamates, sulfonamides, etc.

In certain embodiments, the linker group(s) of the present inventionrepresent a hydrocarbylene group wherein one or more methylene groups isoptionally replaced by a group Y (provided that none of the Y groups areadjacent to each other), wherein each Y, independently for eachoccurrence, is selected from, substituted or unsubstituted aryl,heteroaryl, cycloalkyl, heterocycloalkyl, or —O—, C(═X) (wherein X isNR₁, O or S), —OC(O)—, —C(═O)O, —NR₁—, —NR₁CO—, —C(O)NR₁—, —S(O)_(n)—(wherein n is 0, 1, or 2), —OC(O)—NR₁, —NR₁—C(O)—NR₁—, —NR₁—C(NR₁)—NR₁—,and —B(OR₁)—; and R₁, independently for each occurrence, represents H ora lower alkyl.

In certain embodiments, the linker group represents a derivatized ornon-derivatized amino acid. In certain embodiments, linker groups withone or more terminal carboxyl groups may be conjugated to the polymer.In certain embodiments, one or more of these terminal carboxyl groupsmay be capped by covalently attaching them to a therapeutic agent, atargeting moiety, or a cyclodextrin moiety via an (thio)ester or amidebond. In still other embodiments, linker groups with one or moreterminal hydroxyl, thiol, or amino groups may be incorporated into thepolymer. In preferred embodiments, one or more of these terminalhydroxyl groups may be capped by covalently attaching them to atherapeutic agent, a targeting moiety, or a cyclodextrin moiety via an(thio)ester, amide, carbonate, carbamate, thiocarbonate, orthiocarbamate bond. In certain embodiments, these (thio)ester, amide,(thio)carbonate or (thio)carbamates bonds may be biohydrolyzable, i.e.,capable of being hydrolyzed under biological conditions.

In certain embodiments, the polymers as described above havepolydispersities less than about 3, or even less than about 2.

In certain embodiments, the therapeutic agent is a small molecule, apeptide, a protein, or a polymer that has therapeutic function. Incertain embodiments, the agent is an anti-cancer (such as camptothecinor related derivatives), anti-fungal, anti-bacterial, anti-mycotic, oranti-viral therapeutic. In certain embodiments, the agent is a receptoragonist. In certain embodiments, the agent is a receptor antagonist. Incertain embodiments, the therapeutic agent is a protease inhibitor.Furthermore, a polymer of the present invention may contain one kind oftherapeutic agent, or may contain more than one kind of therapeuticagent. For instance, two or more different cancer drugs, or a cancerdrug and an immunosuppressant, or an antibiotic and an anti-inflammatoryagent may be grafted on to the polymer via optional linkers. Byselecting different linkers for different drugs, the release of eachdrug may be attenuated to achieve maximal dosage and efficacy.

One embodiment of the present invention provides an improved delivery ofcertain hydrophobic small molecule therapeutics by covalentlyconjugating them to cyclodextrin containing polymers. Such conjugationimproves the aqueous solubility and hence the bioavailability of thetherapeutic agents. Accordingly, in one embodiment of the invention, thetherapeutic agent is a hydrophobic compound with a logP>0.4, >0.6, >0.8, >1, >2, >3, >4, or even >5. In other embodiments, ahydrophobic therapeutic agent, such as camptothecin, may be conjugatedto another compound, such as an amino acid, prior to covalentlyattaching the conjugate on to the polymer. Examples of amino acidderivatized camptothecin molecules are illustrated in Scheme V.

The polymer conjugates of the present invention preferably havemolecular weights in the range of 10,000 to 500,000; 30,000 to 200,000;or even 70,000 to 150,000 amu.

In certain embodiments, the cyclodextrin moieties make up at least about2%, 5% or 10% by weight, up to 20%, 30%, 50% or even 80% of thecyclodextrin-modified polymer by weight. In certain embodiments, thetherapeutic agents, or targeting ligands make up at least about 1%, 5%,10% or 15%, 20%, 25%, 30% or even 35% of the cyclodextrin-modifiedpolymer by weight. Number-average molecular weight (M_(n)) may also varywidely, but generally fall in the range of about 1,000 to about 500,000daltons, preferably from about 5000 to about 200,000 daltons and, evenmore preferably, from about 10,000 to about 100,000. Most preferably,M_(n) varies between about 12,000 and 65,000 daltons. In certain otherembodiments, M_(n) varies between about 3000 and 150,000 daltons. Withina given sample of a subject polymer, a wide range of molecular weightsmay be present. For example, molecules within the sample may havemolecular weights that differ by a factor of 2, 5, 10, 20, 50, 100, ormore, or that differ from the average molecular weight by a factor of 2,5, 10, 20, 50, 100, or more. Exemplary cyclodextrin moieties includecyclic structures consisting essentially of from 7 to 9 saccharidemoieties, such as cyclodextrin and oxidized cyclodextrin. A cyclodextrinmoiety optionally comprises a linker moiety that forms a covalentlinkage between the cyclic structure and the polymer backbone,preferably having from 1 to 20 atoms in the chain, such as alkyl chains,including dicarboxylic acid derivatives (such as glutaric acidderivatives, succinic acid derivatives, and the like), and heteroalkylchains, such as oligoethylene glycol chains.

Cyclodextrins are cyclic polysaccharides containing naturally occurringD-(+)-glucopyranose units in an α-(1,4) linkage. The most commoncyclodextrins are alpha ((α)-cyclodextrins, beta (β)-cyclodextrins andgamma (γ)-cyclodextrins which contain, respectively six, seven, or eightglucopyranose units. Structurally, the cyclic nature of a cyclodextrinforms a torus or donut-like shape having an inner apolar or hydrophobiccavity, the secondary hydroxyl groups situated on one side of thecyclodextrin torus and the primary hydroxyl groups situated on theother. Thus, using (β)-cyclodextrin as an example, a cyclodextrin isoften represented schematically as follows.

The side on which the secondary hydroxyl groups are located has a widerdiameter than the side on which the primary hydroxyl groups are located.The present invention contemplates covalent linkages to cyclodextrinmoieties on the primary and/or secondary hydroxyl groups. Thehydrophobic nature of the cyclodextrin inner cavity allows forhost-guest inclusion complexes of a variety of compounds, e.g.,adamantane. (Comprehensive Supramolecular Chemistry, Volume 3, J. L.Atwood et al., eds., Pergamon Press (1996); T. Cserhati, AnalyticalBiochemistry, 225:328-332(1995); Husain et al., Applied Spectroscopy,46:652-658 (1992); FR 2 665 169). Additional methods for modifyingpolymers are disclosed in Suh, J. and Noh, Y., Bioorg. Med. Chem. Lett.1998, 8, 1327-1330.

In certain embodiments, the present invention contemplates linear,water-soluble, cyclodextrin-containing polymer, wherein a plurality ofbioactive moieties are covalently attached to the polymer throughattachments that are cleaved under biological conditions to release thebioactive moieties, wherein administration of the polymer to a patientresults in release of the bioactive agent over a period of at least 2,3, 5, 6, 8, 10, 15, 20, 24, 36, 48 or even 72 hours.

In certain embodiments, the present invention contemplates attenuatingthe rate of release of the therapeutic agent by introducing variouslinking groups between the therapeutic agent and/or targeting ligand andthe polymer. Thus, in certain embodiments, the polymeric therapeutics ofthe present invention are compositions for controlled delivery oftherapeutic agents. One skilled in the art would also recognize that bylabeling the therapeutic agent and/or targeting ligand with radionuclei,or by forming complexes of NMR active nuclei, e.g., technetium,gadolinium, or dysprosium, the polymers of the present invention canachieve a dual diagnostic/therapeutic utility.

In other embodiments, the polymeric compounds stabilize the bioactiveform of a therapeutic agent which exists in equilibrium between anactive and inactive form. For instance, conjugating the therapeuticagent to the polymers of the present invention may shift the equilibriumbetween two tautomeric forms of the agent to the bioactive tautomer. Inother embodiment, the polymeric compounds may attenuate the equilibriumbetween lactonic and acid forms of a therapeutic agent.

One method to determine molecular weight is by gel permeationchromatography (“GPC”), e.g., mixed bed columns, CH₂Cl₂ solvent, lightscattering detector, and off-line dn/dc. Other methods are known in theart.

In other embodiments, the polymer conjugate of the invention may be aflexible or flowable material. When the polymer used is itself flowable,the polymer composition of the invention, even when viscous, need notinclude a biocompatible solvent to be flowable, although trace orresidual amounts of biocompatible solvents may still be present.

While it is possible that the biodegradable polymer or the biologicallyactive agent may be dissolved in a small quantity of a solvent that isnon-toxic to more efficiently produce an amorphous, monolithicdistribution or a fine dispersion of the biologically active agent inthe flexible or flowable composition, it is an advantage of theinvention that, in a preferred embodiment, no solvent is needed to forma flowable composition. Moreover, the use of solvents is preferablyavoided because, once a polymer composition containing solvent is placedtotally or partially within the body, the solvent dissipates or diffusesaway from the polymer and must be processed and eliminated by the body,placing an extra burden on the body's clearance ability at a time whenthe illness (and/or other treatments for the illness) may have alreadydeleteriously affected it.

However, when a solvent is used to facilitate mixing or to maintain theflowability of the polymer conjugate of the invention, it should benon-toxic, otherwise biocompatible, and should be used in relativelysmall amounts. Solvents that are toxic should not be used in anymaterial to be placed even partially within a living body. Such asolvent also must not cause substantial tissue irritation or necrosis atthe site of administration.

Examples of suitable biocompatible solvents, when used, includeN-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol,acetone, methyl acetate, ethyl acetate, methyl ethyl ketone,dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam,oleic acid, or 1-dodecylazacylcoheptanone. Preferred solvents includeN-methylpyrrolidone, 2-pyrrolidone, dimethylsulfoxide, and acetonebecause of their solvating ability and their biocompatibility.

In certain embodiments, the subject polymer conjugates are soluble inone or more common organic solvents for ease of fabrication andprocessing. Common organic solvents include such solvents as chloroform,dichloromethane, dichloroethane, 2-butanone, butyl acetate, ethylbutyrate, acetone, ethyl acetate, dimethylacetamide,N-methylpyrrolidone, dimethylformamide, and dimethylsulfoxide.

One aspect of the present invention contemplates attaching a hydrophobictherapeutic agent such as (S)-20-camptothecin to linear or branchedcyclodextrin-containing polymers for better delivery of the drug.(S)-20-camptothecin (CPT), an alkaloid isolated from Camptithecaaccuminata in the late 1950's, was found to exhibit anticancer activityby inhibiting the action of topoisomerase I during the S-phase of thecell cycle. Its application in human cancer treatment, however, islimited due to several factors, especially its undesirable interactionswith human serum albumin, instability of the bioactive lactone form, andpoor aqueous solubility. In order to circumvent this problem, many CPTanalogs have been developed to improve lactone stability and aqueoussolubility. Topotecan and irinotecan are analogs of CPT that havealready been approved by FDA for human cancer treatment. The presentinvention discloses various types of linear, branched, or graftedcyclodextrin-containing polymers wherein (S)-20-camptothecin iscovalently bound to the polymer. In certain embodiments, the drug iscovalently linked via a biohydrolyzable bond selected from an ester,amide, carbamates, or carbonate.

An exemplary synthetic scheme for covalently bonding a derivatized CD to20(S)-camptothecin is shown in Scheme I.

Without intending to limit the scope of the invention, a generalstrategy for synthesizing linear, branched or graftedcyclodextrin-containing polymers (CD Polymer) for loading a therapeuticagent such as camptothecin, and an optional targeting ligand is shown inFIG. 13.

To illustrate further, without intending to be limiting, comonomer Aprecursors, cyclodextrin moieties, therapeutic agents, and/or targetingligands may be assembled as shown in Schemes IIa-IIb (FIGS. 14 and 15).Note that in schemes IIa-b, in any given reaction there may be more thanone comonomer A precursor, cyclodextrin moiety, therapeutic agent ortargeting ligand that is of the same type or different. Furthermore,prior to polymerization, one or more comonomer A precursor, cyclodextrinmoiety, therapeutic agent or targeting ligand may be covalently linkedwith each other in one or more separate step.

Scheme IIa: General scheme for graft polymers. The comonomer Aprecursor, cyclodextrin moiety, therapeutic agent and targeting ligandare as defined above. Furthermore, one skilled in the art may choosefrom a variety of reactive groups, e.g., hydroxyls, carboxyls, halides,amines, and activated ethenes, ethynes, or aromatic groups in orderachieve polymerization. For further examples of reactive groups aredisclosed in Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th Edition, 2000.

Scheme IIb: General scheme of preparing linear cyclodextrin-containingpolymers. One skilled in the art would recognize that by choosing acomonomer A precursor that has multiple reactive groups polymerbranching can be achieved.

Examples for different ways of synthesizing linear cyclodextrin-CPTpolymers are shown in Schemes III-VIII.

Examples for grafting cyclodextrins on to side-chains of CPT-containingpolymers subunits are shown in Schemes IX-XII. Each subunit may repeatany number of times, and one subunit may occur with substantially thesame frequency, more often, or less often than another subunit, suchthat both subunits may be present in approximately the same amount, orin differing amounts, which may differ slightly or be highly disparate,e.g., one subunit is present nearly to the exclusion of the other.

In certain instances, the polymers are random copolymers, in which thedifferent subunits and/or other monomeric units are distributed randomlythroughout the polymer chain. Thus, where the formula X_(m)—Y_(n)—Z_(o)appears, wherein X, Y and Z are polymer subunits, these subunits may berandomly interspersed throughout the polymer backbone. In part, the term“random” is intended to refer to the situation in which the particulardistribution or incorporation of monomeric units in a polymer that hasmore than one type of monomeric units is not directed or controlleddirectly by the synthetic protocol, but instead results from featuresinherent to the polymer system, such as the reactivity, amounts ofsubunits and other characteristics of the synthetic reaction or othermethods of manufacture, processing, or treatment.

The present invention further contemplates CD-polymers synthesized usingCD-biscysteine monomer and a di-NHS ester such as PEG-DiSPA or PEG-BTCas shown in Schemes XIII-XIV.

In certain embodiments, the present invention discloses severalstrategies to increase drug loading as shown in FIG. 1.

(b) Targeting Ligand

As mentioned above, one aspect of the present invention contemplatesattaching a therapeutic agent to the polymer conjugates describedherein.

In certain embodiments, the polymer conjugate further comprises atargeting ligand. Thus in certain embodiments, a receptor, cell, and/ortissue-targeting ligand, or a precursor thereof is coupled to a polymerconjugate. As used herein the term “targeting ligand” refers to anymaterial or substance which may promote targeting of receptors, cells,and/or tissues in vivo or in vitro with the compositions of the presentinvention. The targeting ligand may be synthetic, semi-synthetic, ornaturally-occurring. Materials or substances which may serve astargeting ligands include, for example, proteins, including antibodies,antibody fragments, hormones, hormone analogues, glycoproteins andlectins, peptides, polypeptides, amino acids, sugars, saccharides,including monosaccharides and polysaccharides, carbohydrates, smallmolecules, vitamins, steroids, steroid analogs, hormones, cofactors,bioactive agents, and genetic material, including nucleosides,nucleotides, nucleotide acid constructs and polynucleotides. As usedherein, the term “precursor” to a targeting ligand refers to anymaterial or substance which may be converted to a targeting ligand. Suchconversion may involve, for example, anchoring a precursor to atargeting ligand. Exemplary targeting precursor moieties includemaleimide groups, disulfide groups, such as ortho-pyridyl disulfide,vinylsulfone groups, azide groups, and α-iodo acetyl groups. Theattachment of the targeting ligand or precursor thereof to the polymermay be accomplished in various ways including but not limited tochelation, covalent attachment, or formation of host-guest complexes. Incertain embodiments, an optional linker group may be present between thetargeting ligand or precursor thereof and the polymer, wherein thelinker group is attached to the polymer via chelation, covalentattachment or form host guest complexes. For example, the one terminalend of a linker group may be attached to the targeting ligand while theother may be attached to an adamantane group, or other such hydrophobicmoiety, which forms a host guest complex with a cyclodextrin moiety.Thus the targeting ligand may be attached to a grafted cyclodextrinmoiety, to a cyclodextrin moiety within the polymeric chain, or to thepolymeric chain itself. The number of targeting ligands per polymericchain may vary according to various factors including but not limited tothe identity of the therapeutic agent, nature of the disease, type ofpolymer chain. Structures of possible linker groups are the same aslinker groups defined elsewhere in this application.

(c) Pharmaceutical Compositions, Formulations and Dosages

In part, a biocompatible polymer composition of the present inventionincludes a biocompatible and optionally biodegradable polymer, such asone having the recurring monomeric units shown in one of the foregoingformulas, optionally including any other biocompatible and optionallybiodegradable polymer mentioned above or known in the art. In certainembodiments, the compositions are non-pyrogenic, e.g., do not triggerelevation of a patient's body temperature by more than a clinicallyacceptable amount.

The subject compositions may contain a “drug”, “therapeutic agent,”“medicament,” or “bioactive substance,” which are biologically,physiologically, or pharmacologically active substances that act locallyor systemically in the human or animal body. For example, a subjectcomposition may include any of the other compounds discussed above.

Various forms of the medicaments or biologically active materials may beused which are capable of being released from the polymer matrix intoadjacent tissues or fluids. They may be hydrophobic molecules, neutralmolecules, polar molecules, or molecular complexes capable of hydrogenbonding. They may be in the form of ethers, esters, amides and the like,including prodrugs which are biologically activated when injected intothe human or animal body, e.g., by cleavage of an ester or amide. Atherapeutic agent in a subject composition may vary widely with thepurpose for the composition.

Plasticizers and stabilizing agents known in the art may be incorporatedin polymers of the present invention. In certain embodiments, additivessuch as plasticizers and stabilizing agents are selected for theirbiocompatibility. In certain embodiments, the additives are lungsurfactants, such as 1,2-dipalmitoylphosphatidycholine (DPPC) andL-α-phosphatidylcholine (PC).

A composition of this invention may further contain one or more adjuvantsubstances, such as fillers, thickening agents or the like. In otherembodiments, materials that serve as adjuvants may be associated withthe polymer matrix. Such additional materials may affect thecharacteristics of the polymer matrix that results.

For example, fillers, such as bovine serum albumin (BSA) or mouse serumalbumin (MSA), may be associated with the polymer matrix. In certainembodiments, the amount of filler may range from about 0.1 to about 50%or more by weight of the polymer matrix, or about 2.5, 5, 10, 25, or 40percent. Incorporation of such fillers may affect the biodegradation ofthe polymeric material and/or the sustained release rate of anyencapsulated substance. Other fillers known to those of skill in theart, such as carbohydrates, sugars, starches, saccharides, cellulosesand polysaccharides, including mannitose and sucrose, may be used incertain embodiments of the present invention.

In other embodiments, spheronization enhancers facilitate the productionof subject polymeric matrices that are generally spherical in shape.Substances such as zein, microcrystalline cellulose or microcrystallinecellulose co-processed with sodium carboxymethyl cellulose may conferplasticity to the subject compositions as well as implant strength andintegrity. In particular embodiments, during spheronization, extrudatesthat are rigid, but not plastic, result in the formation of dumbbellshaped implants and/or a high proportion of fines, and extrudates thatare plastic, but not rigid, tend to agglomerate and form excessivelylarge implants. In such embodiments, a balance between rigidity andplasticity is desirable. The percent of spheronization enhancer in aformulation typically range from 10 to 90% (w/w).

In certain embodiments, a subject composition includes an excipient. Aparticular excipient may be selected based on its melting point,solubility in a selected solvent (e.g., a solvent that dissolves thepolymer and/or the therapeutic agent), and the resulting characteristicsof the microparticles.

Excipients may comprise a few percent, about 5%, 10%, 15%, 20%, 25%,30%, 40%, 50%, or higher percentage of the subject compositions.

Buffers, acids and bases may be incorporated in the subject compositionsto adjust their pH. Agents to increase the diffusion distance of agentsreleased from the polymer matrix may also be included.

Disintegrants are substances that, in the presence of liquid, promotethe disruption of the subject compositions. Disintegrants are most oftenused in implants, in which the function of the disintegrant is tocounteract or neutralize the effect of any binding materials used in thesubject formulation. In general, the mechanism of disintegrationinvolves moisture absorption and swelling by an insoluble material.

Examples of disintegrants include croscarmellose sodium and crospovidonewhich, in certain embodiments, may be incorporated into the polymericmatrices in the range of about 1-20% of total matrix weight. In othercases, soluble fillers such as sugars (mannitol and lactose) may also beadded to facilitate disintegration of implants.

Other materials may be used to advantage or to control the desiredrelease rate of a therapeutic agent for a particular treatment protocol.For example, if the sustained release is too slow for a particularapplication, a pore-forming agent may be added to generate additionalpores in the matrix. Any biocompatible water-soluble material may beused as the pore-forming agent. They may be capable of dissolving,diffusing or dispersing out of the formed polymer system whereupon poresand microporous channels are generated in the system. The amount ofpore-forming agent (and size of dispersed particles of such pore-formingagent, if appropriate) within the composition should affect the size andnumber of the pores in the polymer system.

Pore-forming agents include any pharmaceutically acceptable organic orinorganic substance that is substantially miscible in water and bodyfluids and will dissipate from the forming and formed matrix intoaqueous medium or body fluids or water-immiscible substances thatrapidly degrade to water-soluble substances.

Suitable pore-forming agents include, for example, sugars such assucrose and dextrose, salts such as sodium chloride and sodiumcarbonate, and polymers such as hydroxylpropylcellulose,carboxymethylcellulose, polyethylene glycol, and PVP. The size andextent of the pores may be varied over a wide range by changing themolecular weight and percentage of pore-forming agent incorporated intothe polymer system.

The charge, lipophilicity or hydrophilicity of any subject polymericmatrix may be modified by attaching in some fashion an appropriatecompound to the surface of the matrix. For example, surfactants may beused to enhance wettability of poorly soluble or hydrophobiccompositions. Examples of suitable surfactants include dextran,polysorbates and sodium lauryl sulfate. In general, surfactants are usedin low concentrations, generally less than about 5%.

Binders are adhesive materials that may be incorporated in polymericformulations to bind and maintain matrix integrity. Binders may be addedas dry powder or as solution. Sugars and natural and synthetic polymersmay act as binders.

Materials added specifically as binders are generally included in therange of about 0.5%-15% w/w of the matrix formulation. Certainmaterials, such as microcrystalline cellulose, also used as aspheronization enhancer, also have additional binding properties.

Various coatings may be applied to modify the properties of thematrices.

Three exemplary types of coatings are seal, gloss and enteric coatings.Other types of coatings having various dissolution or erosion propertiesmay be used to further modify subject matrices behavior, and suchcoatings are readily known to one of ordinary skill in the art.

The seal coat may prevent excess moisture uptake by the matrices duringthe application of aqueous based enteric coatings. The gloss coatgenerally improves the handling of the finished matrices. Water-solublematerials such as hydroxypropylcellulose may be used to seal coat andgloss coat implants. The seal coat and gloss coat are generally sprayedonto the matrices until an increase in weight between about 0.5% andabout 5%, often about 1% for a seal coat and about 3% for a gloss coat,has been obtained.

Enteric coatings consist of polymers which are insoluble in the low pH(less than 3.0) of the stomach, but are soluble in the elevated pH(greater than 4.0) of the small intestine. Polymers such as EUDRAGIT™,RohmTech, Inc., Malden, Mass., and AQUATERIC™, FMC Corp., Philadelphia,Pa., may be used and are layered as thin membranes onto the implantsfrom aqueous solution or suspension or by a spray drying method. Theenteric coat is generally sprayed to a weight increase of about 1% toabout 30%, preferably about 10 to about 15% and may contain coatingadjuvants such as plasticizers, surfactants, separating agents thatreduce the tackiness of the implants during coating, and coatingpermeability adjusters.

The present compositions may additionally contain one or more optionaladditives such as fibrous reinforcement, colorants, perfumes, rubbermodifiers, modifying agents, etc. In practice, each of these optionaladditives should be compatible with the resulting polymer and itsintended use. Examples of suitable fibrous reinforcement include PGAmicrofibrils, collagen microfibrils, cellulosic microfibrils, andolefinic microfibrils. The amount of each of these optional additivesemployed in the composition is an amount necessary to achieve thedesired effect.

The therapeutic polymer conjugates as described herein can beadministered in various pharmaceutical formulations, depending on thedisorder to be treated and the age, condition and body weight of thepatient, as is well known in the art. For example, where the compoundsare to be administered orally, they may be formulated as tablets,capsules, granules, powders or syrups; or for parenteral administration,they may be formulated as injections (intravenous, intramuscular orsubcutaneous), drop infusion preparations or suppositories. Forapplication by the ophthalmic mucous membrane route, they may beformulated as eyedrops or eye ointments. These formulations can beprepared by conventional means, and, if desired, the active ingredientmay be mixed with any conventional additive, such as an excipient, abinder, a disintegrating agent, a lubricant, a corrigent, a solubilizingagent, a suspension aid, an emulsifying agent or a coating agent.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of thetherapeutic agent is recommended for an adult human patient, and thismay be administered in a single dose or in divided doses.

The precise time of administration and/or amount of therapeutic polymerconjugate that will yield the most effective results in terms ofefficacy of treatment in a given patient will depend upon the activity,pharmacokinetics, and bioavailability of a particular compound,physiological condition of the patient (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosageand type of medication), route of administration, etc. However, theabove guidelines can be used as the basis for fine-tuning the treatment,e.g., determining the optimum time and/or amount of administration,which will require no more than routine experimentation consisting ofmonitoring the subject and adjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose therapeutic polymer conjugates, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the therapeuticpolymer conjugates. These salts can be prepared in situ during the finalisolation and purification of the therapeutic polymer conjugates, or byseparately reacting a purified polymer in its free base form with asuitable organic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

In other cases, the therapeutic polymer conjugates useful in the methodsof the present invention may contain one or more acidic functionalgroups and, thus, are capable of forming pharmaceutically acceptablesalts with pharmaceutically acceptable bases. The term “pharmaceuticallyacceptable salts” in these instances refers to the relatively non-toxic,inorganic and organic base addition salts of the polymer(s). These saltscan likewise be prepared in situ during the final isolation andpurification of the polymer(s), or by separately reacting the purifiedpolymer(s) in its free acid form with a suitable base, such as thehydroxide, carbonate or bicarbonate of a pharmaceutically acceptablemetal cation, with ammonia, or with a pharmaceutically acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including ophthalmic, otic,buccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally, out ofone hundred percent, this amount will range from about 1 percent toabout ninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a therapeutic polymer conjugate(s) with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation a therapeutic polymer conjugate with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, gums, lozenges (using a flavoredbasis, usually sucrose and acacia or tragacanth), powders, granules, oras a solution or a suspension in an aqueous or non-aqueous liquid, or asan oil-in-water or water-in-oil liquid emulsion, or as an elixir orsyrup, or as pastilles (using an inert base, such as gelatin andglycerin, or sucrose and acacia) and/or as mouthwashes and the like,each containing a predetermined amount of a therapeutic polymerconjugate(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active therapeutic polymer conjugatesmay contain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more therapeuticpolymer conjugates with one or more suitable nonirritating excipients orcarriers comprising for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of atherapeutic polymer conjugate(s) include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive component may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition toligand(s), excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to a therapeutic polymerconjugate(s), excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The therapeutic polymer conjugate(s) can be alternatively administeredby aerosol. This is accomplished by preparing an aqueous aerosol,liposomal preparation or solid particles containing the compound. Anonaqueous (e.g., fluorocarbon propellant) suspension could be used.Sonic nebulizers are preferred because they minimize exposing the agentto shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a therapeutic polymer conjugate(s) to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe ligand across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing thepeptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more therapeutic polymer conjugate(s) incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices oftherapeutic polymer conjugate(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the therapeutic polymer conjugate(s) of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are of course given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a therapeutic polymer conjugate, drugor other material other than directly into the central nervous system,such that it enters the patient's system and, thus, is subject tometabolism and other like processes, for example, subcutaneousadministration.

These therapeutic polymer conjugate(s) may be administered to humans andother animals for therapy by any suitable route of administration,including orally, nasally, as by, for example, a spray, rectally,intravaginally, parenterally, intracisternally and topically, as bypowders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the therapeuticpolymer conjugate(s), which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

(c) Physical Structures of the Subject Compositions

The subject polymers may be formed in a variety of shapes. For example,in certain embodiments, subject polymer matrices may be presented in theform of microparticles or nanoparticles. Microspheres typically comprisea biodegradable polymer matrix incorporating a drug. Microspheres can beformed by a wide variety of techniques known to those of skill in theart. Examples of microsphere forming techniques include, but are notlimited to, (a) phase separation by emulsification and subsequentorganic solvent evaporation (including complex emulsion methods such asoil in water emulsions, water in oil emulsions and water-oil-wateremulsions); (b) coacervation-phase separation; (c) melt dispersion; (d)interfacial deposition; (e) in situ polymerization; (f) spray drying andspray congealing; (g) air suspension coating; and (h) pan and spraycoating. These methods, as well as properties and characteristics ofmicrospheres are disclosed in, for example, U.S. Pat. Nos. 4,652,441;5,100,669; 4,526,938; WO 93/24150; EPA 0258780 A2; U.S. Pat. Nos.4,438,253; and 5,330,768, the entire disclosures of which areincorporated by reference herein.

To prepare microspheres of the present invention, several methods can beemployed depending upon the desired application of the deliveryvehicles. Suitable methods include, but are not limited to, spraydrying, freeze drying, air drying, vacuum drying, fluidized-bed drying,milling, co-precipitation and critical fluid extraction. In the case ofspray drying, freeze drying, air drying, vacuum drying, fluidized-beddrying and critical fluid extraction; the components (stabilizingpolyol, bioactive material, buffers, etc.) are first dissolved orsuspended in aqueous conditions. In the case of milling, the componentsare mixed in the dried form and milled by any method known in the art.In the case of co-precipitation, the components are mixed in organicconditions and processed as described below. Spray drying can be used toload the stabilizing polyol with the bioactive material. The componentsare mixed under aqueous conditions and dried using precision nozzles toproduce extremely uniform droplets in a drying chamber. Suitable spraydrying machines include, but are not limited to, Buchi, NIRO, APV andLab-plant spray driers used according to the manufacturer'sinstructions.

The shape of microparticles and nanoparticles may be determined byscanning electron microscopy. Spherically shaped nanoparticles are usedin certain embodiments, for circulation through the bloodstream. Ifdesired, the particles may be fabricated using known techniques intoother shapes that are more useful for a specific application.

In addition to intracellular delivery of a therapeutic agent, it alsopossible that particles of the subject compositions, such asmicroparticles or nanoparticles, may undergo endocytosis, therebyobtaining access to the cell. The frequency of such an endocytosisprocess will likely depend on the size of any particle.

In certain embodiments, solid articles useful in defining shape andproviding rigidity and structural strength to the polymeric matrices maybe used. For example, a polymer may be formed on a mesh or other weavefor implantation. A polymer may also be fabricated as a stent or as ashunt, adapted for holding open areas within body tissues or fordraining fluid from one body cavity or body lumen into another. Further,a polymer may be fabricated as a drain or a tube suitable for removingfluid from a post-operative site, and in some embodiments adaptable foruse with closed section drainage systems such as Jackson-Pratt drainsand the like as are familiar in the art.

The mechanical properties of the polymer may be important for theprocessability of making molded or pressed articles for implantation.For example, the glass transition temperature may vary widely but mustbe sufficiently lower than the temperature of decomposition toaccommodate conventional fabrication techniques, such, as compressionmolding, extrusion, or injection molding.

(d) Biodegradability and Release Characteristics

In certain embodiments, the polymers and blends of the presentinvention, upon contact with body fluids, undergo gradual degradation.The life of a biodegradable polymer in vivo depends upon, among otherthings, its molecular weight, crystallinity, biostability, and thedegree of crosslinking. In general, the greater the molecular weight,the higher the degree of crystallinity, and the greater the biostability, the slower biodegradation will be.

If a subject composition is formulated with a therapeutic agent or othermaterial, release of such an agent or other material for a sustained orextended period as compared to the release from an isotonic salinesolution generally results. Such release profile may result in prolongeddelivery (over, say 1 to about 2,000 hours, or alternatively about 2 toabout 800 hours) of effective amounts (e.g., about 0.0001 mg/kg/hour toabout 10 mg/kg/hour) of the agent or any other material associated withthe polymer.

A variety of factors may affect the desired rate of hydrolysis ofpolymers of the subject invention, the desired softness and flexibilityof the resulting solid matrix, rate and extent of bioactive materialrelease. Some of such factors include the selection/identity of thevarious subunits, the enantiomeric or diastereomeric purity of themonomeric subunits, homogeneity of subunits found in the polymer, andthe length of the polymer. For instance, the present inventioncontemplates heteropolymers with varying linkages, and/or the inclusionof other monomeric elements in the polymer, in order to control, forexample, the rate of biodegradation of the matrix.

To illustrate further, a wide range of degradation rates may be obtainedby adjusting the hydrophobicities of the backbones or side chains of thepolymers while still maintaining sufficient biodegradability for the useintended for any such polymer. Such a result may be achieved by varyingthe various functional groups of the polymer. For example, thecombination of a hydrophobic backbone and a hydrophilic linkage producesheterogeneous degradation because cleavage is encouraged whereas waterpenetration is resisted.

One protocol generally accepted in the field that may be used todetermine the release rate of any therapeutic agent or other materialloaded in the polymer matrices of the present invention involvesdegradation of any such matrix in a 0.1 M PBS solution (pH 7.4) at 37°C., an assay known in the art. For purposes of the present invention,the term “PBS protocol” is used herein to refer to such protocol.

In certain instances, the release rates of different polymer systems ofthe present invention may be compared by subjecting them to such aprotocol. In certain instances, it may be necessary to process polymericsystems in the same fashion to allow direct and relatively accuratecomparisons of different systems to be made. For example, the presentinvention teaches several different means of formulating the polymeric,matrices of the present invention. Such comparisons may indicate thatany one polymeric system releases incorporated material at a rate fromabout 2 or less to about 1000 or more times faster than anotherpolymeric system.

Alternatively, a comparison may reveal a rate difference of about 3, 5,7, 10, 25, 50, 100, 250, 500 or 750 times. Even higher rate differencesare contemplated by the present invention and release rate protocols.

In certain embodiments, when formulated in a certain manner, the releaserate for polymer systems of the present invention may present as mono-or bi-phasic.

Release of any material incorporated into the polymer matrix, which isoften provided as a microsphere, may be characterized in certaininstances by an initial increased release rate, which may release fromabout 5 to about 50% or more of any incorporated material, oralternatively about 10, 15, 20, 25, 30 or 40%, followed by a releaserate of lesser magnitude.

The release rate of any incorporated material may also be characterizedby the amount of such material released per day per mg of polymermatrix. For example, in certain embodiments, the release rate may varyfrom about 1 ng or less of any incorporated material per day per mg ofpolymeric system to about 500 or more ng/day/mg. Alternatively, therelease rate may be about 0.05, 0.5, 5, 10, 25, 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, or 500 ng/day/mg. In still otherembodiments, the release rate of any incorporated material may be 10,000ng/day/mg, or even higher. In certain instances, materials incorporatedand characterized by such release rate protocols may include therapeuticagents, fillers, and other substances.

In another aspect, the rate of release of any material from any polymermatrix of the present invention may be presented as the half-life ofsuch material in the matrix.

In addition to the embodiment involving protocols for in vitrodetermination of release rates, in vivo protocols, whereby in certaininstances release rates for polymeric systems may be determined in vivo,are also contemplated by the present invention. Other assays useful fordetermining the release of any material from the polymers of the presentsystem are known in the art.

(e) Implants and Delivery Systems

In its simplest form, a biodegradable delivery system for a therapeuticagent consists of a dispersion of such a therapeutic agent in a polymermatrix. In other embodiments, an article is used for implantation,injection, or otherwise placed totally or partially within the body, thearticle comprising the subject compositions. It is particularlyimportant that such an article result in minimal tissue irritation whenimplanted or injected into vasculated tissue.

Biodegradable delivery systems, and articles thereof, may be prepared ina variety of ways known in the art. The subject polymer may bemelt-processed using conventional extrusion or injection moldingtechniques, or these products may be prepared by dissolving in anappropriate solvent, followed by formation of the device, and subsequentremoval of the solvent by evaporation or extraction.

Once a system or implant article is in place, it should remain in atleast partial contact with a biological fluid, such as blood, internalorgan secretions, mucus membranes, cerebrospinal fluid, and the like toallow for sustained release of any encapsulated therapeutic agent.

(f) Methods of Manufacturing

Generally, compounds of the present invention can be prepared in one oftwo ways: monomers bearing therapeutic agents, targeting ligands, and/orcyclodextrin moieties can be polymerized, or polymer backbones can bederivatized with therapeutic agents, targeting ligands, and/orcyclodextrin moieties.

Thus, in one embodiment, the present invention contemplates thesynthesis of compounds of the invention by reacting monomers M-L-CD andM-L-D (and, optionally, M-L-T), wherein

CD represents a cyclic moiety, such as a cyclodextrin molecule, orderivative thereof;

L, independently for each occurrence, may be absent or represents alinker group;

D, independently for each occurrence, represents the same or differenttherapeutic agent or prodrugs thereof;

T, independently for each occurrence, represents the same or differenttargeting ligand or precursor thereof; and

M represents a monomer subunit bearing one or more reactive moietiescapable of undergoing a polymerization reaction with one or more other Min the monomers in the reaction mixture, under conditions that causepolymerization of the monomers to take place.

In certain embodiments, the reaction mixture may further comprisemonomers that do not bear CD, T, or D moieties, e.g., to space thederivatized monomer units throughout the polymer.

In an alternative embodiment, the invention contemplates synthesizing acompound of the present invention by reacting a polymer P (the polymerbearing a plurality of reactive groups, such as carboxylic acids,alcohols, thiols, amines, epoxides, etc.) with grafting agents X-L-CDand Y-L-D (and, optionally, Z-L-T), wherein

CD represents a cyclic moiety, such as a cyclodextrin molecule, orderivative thereof;

L, independently for each occurrence, may be absent or represents alinker group;

D, independently for each occurrence, represents the same or differenttherapeutic agent or prodrugs thereof;

T, independently for each occurrence, represents the same or differenttargeting ligand or precursor thereof;

X, independently for each occurrence, represents a reactive group, suchas carboxylic acids, alcohols, thiols, amines, epoxides, etc., capableof forming a covalent bond with a reactive group of the polymer; and

Y and Z, independently for each occurrence, represent inclusion hosts orreactive groups, such as carboxylic acids, alcohols, thiols, amines,epoxides, etc., capable of forming a covalent bond with a reactive groupof the polymer or inclusion complexes with CD moieties grafted to thepolymer, under conditions that cause the grafting agents to formcovalent bonds and/or inclusion complexes, as appropriate, with thepolymer or moieties grafted to the polymer.

For example, if the polymer includes alcohols, thiols, or amines asreactive groups, the grafting agents may include reactive groups thatreact with them, such as isocyanates, isothiocyanates, acid chlorides,acid anhydrides, epoxides, ketenes, sulfonyl chlorides, activatedcarboxylic acids (e.g., carboxylic acids treated with an activatingagent such as PyBrOP, carbonyldiimidazole, or another reagent thatreacts with a carboxylic acid to form a moiety susceptible tonucleophilic attack), or other electrophilic moieties known to those ofskill in the art. In certain embodiments, a catalyst may be needed tocause the reaction to take place (e.g., a Lewis acid, a transition metalcatalyst, an amine base, etc.) as will be understood by those of skillin the art.

In certain embodiments, the different grafting agents are reacted withthe polymer simultaneously or substantially simultaneously (e.g., in aone-pot reaction), or are reacted sequentially with the polymer(optionally with a purification and/or wash step between reactions).

Another aspect of the present invention is a method for manufacturingthe linear or branched cyclodextrin-containing polymers represented byformulae I-III. While the discussion below focuses on the preparation oflinear cyclodextrin molecules, one skilled in the art would readilyrecognize that the methods described can be adapted for producingbranched polymers by choosing an appropriate comonomer A precursor.

Accordingly, one embodiment of the invention is a method of preparing alinear cyclodextrin copolymer. According to the invention, a linearcyclodextrin copolymer of the invention may be prepared bycopolymerizing a cyclodextrin monomer precursor disubstituted with anappropriate leaving group with a comonomer A precursor capable ofdisplacing the leaving groups. The leaving group, which may be the sameor different, may be any leaving group known in the art which may bedisplaced upon copolymerization with a comonomer A precursor. In apreferred embodiment, a linear cyclodextrin copolymer may be prepared byiodinating a cyclodextrin monomer precursor to form a diiodinatedcyclodextrin monomer precursor and copolymerizing the diiodinatedcyclodextrin monomer precursor with a comonomer A precursor to form alinear cyclodextrin copolymer having a repeating unit of formula II orIII, or a combination thereof, each as described above. While examplespresented below discuss iodinated cyclodextrin moieties, one skilled inthe art would readily recognize that the present invention contemplatesand encompasses cyclodextrin moieties wherein other leaving groups suchas alkyl and aryl sulfonate may be present instead of iodo groups. In apreferred embodiment, a method of preparing a linear cyclodextrincopolymer of the invention by iodinating a cyclodextrin monomerprecursor as described above to form a diiodinated cyclodextrin monomerprecursor of formula IVa, IVb, IVc or a mixture thereof:

The diiodinated cyclodextrin may be prepared by any means known in theart. (Tabushi et al. J. Am. Chem. 106, 5267-5270 (1984); Tabushi et al.J. Am. Chem. 106, 4580-4584 (1984)). For example, β-cyclodextrin may bereacted with biphenyl-4,4′-disulfonyl chloride in the presence ofanhydrous pyridine to form a biphenyl-4,4′-disulfonyl chloride cappedβ-Cyclodextrin which may then be reacted with potassium iodide toproduce diiodo-β-cyclodextrin. The cyclodextrin monomer precursor isiodinated at only two positions. By copolymerizing the diiodinatedcyclodextrin monomer precursor with a comonomer A precursor, asdescribed above, a linear cyclodextrin polymer having a repeating unitof Formula Ia, Ib, or a combination thereof, also as described above,may be prepared. If appropriate, the iodine or iodo groups may bereplaced with other known leaving groups.

Also according to the invention, the iodo groups or other appropriateleaving group may be displaced with a group that permits reaction with acomonomer A precursor, as described above. For example, a diiodinatedcyclodextrin monomer precursor of formula IVa, IVb, IVc or a mixturethereof may be aminated to form a diaminated cyclodextrin monomerprecursor of formula Va, Vb, Vc or a mixture thereof:

The diaminated cyclodextrin monomer precursor may be prepared by anymeans known in the art. (Tabushi et al. Tetrahedron Lett. 18:11527-1530(1977); Mungall et al., J. Org. Chem. 16591662 (1975)). For example, adiiodo-β-cyclodextrin may be reacted with sodium azide and then reducedto form a diamino-β-cyclodextrin). The cyclodextrin monomer precursor isaminated at only two positions. The diaminated cyclodextrin monomerprecursor may then be copolymerized with a comonomer A precursor, asdescribed above, to produce a linear cyclodextrin copolymer having arepeating unit of formula II-III or a combination thereof, also asdescribed above. However, the amino functionality of a diaminatedcyclodextrin monomer precursor need not be directly attached to thecyclodextrin moiety. Alternatively, the amino functionality or anothernucleophilic functionality may be introduced by displacement of the iodoor other appropriate leaving groups of a cyclodextrin monomer precursorwith amino group containing moieties such as, for example, HSCH₂CH₂NH₂(or a di-nucleophilic molecule more generally represented byHW—(CR₁R₂)_(n)—WH wherein W, independently for each occurrence,represents O, S, or NR₁; R₁ and R₂, independently for each occurrence,represent H, (un)substituted alkyl, (un)substituted aryl,(un)substituted heteroalkyl, (un)substituted heteroaryl) with anappropriate base such as a metal hydride, alkali or alkaline carbonate,or tertiary amine to form a diaminated cyclodextrin monomer precursor offormula Vd, Ve, Vf or a mixture thereof:

A linear oxidized cyclodextrin-containing copolymer of the invention mayalso be prepared by oxidizing a reduced linear cyclodextrin-containingcopolymer of the invention as described below. This method may beperformed as long as the comonomer A does not contain an oxidationsensitive moiety or group such as, for example, a thiol.

According to the invention, a linear cyclodextrin copolymer of theinvention may be oxidized so as to introduce at least one oxidizedcyclodextrin monomer into the copolymer such that the oxidizedcyclodextrin monomer is an integral part of the polymer backbone. Alinear cyclodextrin copolymer which contains at least one oxidizedcyclodextrin monomer is defined as a linear oxidized cyclodextrincopolymer or a linear oxidized cyclodextrin-containing polymer. Thecyclodextrin monomer may be oxidized on either the secondary or primaryhydroxyl side of the cyclodextrin moiety. If more than one oxidizedcyclodextrin monomer is present in a linear oxidized cyclodextrincopolymer of the invention, the same or different cyclodextrin monomersoxidized on either the primary hydroxyl side, the secondary hydroxylside, or both may be present. For illustration purposes, a linearoxidized cyclodextrin copolymer with oxidized secondary hydroxyl groupshas, for example, at least one unit of formula VIa or VIb:

In formulae VIa and VIb, C is a substituted or unsubstituted oxidizedcyclodextrin monomer and A is a comonomer bound, i.e., covalently bound,to the oxidized cyclodextrin C. Also in formulae VIa and VIb, oxidationof the secondary hydroxyl groups leads to ring opening of thecyclodextrin moiety and the formation of aldehyde groups.

A linear oxidized cyclodextrin copolymer may be prepared by oxidation ofa linear cyclodextrin copolymer as discussed above. Oxidation of alinear cyclodextrin copolymer of the invention may be accomplished byoxidation techniques known in the art. (Hisamatsu et al., Starch44:188-191 (1992)). Preferably, an oxidant such as, for example, sodiumperiodate is used. It would be understood by one of ordinary skill inthe art that under standard oxidation conditions that the degree ofoxidation may vary or be varied per copolymer. Thus in one embodiment ofthe invention, a linear oxidized copolymer of the invention may containone oxidized cyclodextrin monomer. In another embodiment, substantiallyall cyclodextrin monomers of the copolymer would be oxidized.

Another method of preparing a linear oxidized cyclodextrin copolymer ofthe invention involves the oxidation of a diiodinated or diaminatedcyclodextrin monomer precursor, as described above, to form an oxidizeddiiodinated or diaminated cyclodextrin monomer precursor andcopolymerization of the oxidized diiodinated or diaminated cyclodextrinmonomer precursor with a comonomer A precursor. In a preferredembodiment, an oxidized diiodinated cyclodextrin monomer precursor offormula VIIa, VIIb, VIIc, or a mixture thereof:

may be prepared by oxidation of a diiodinated cyclodextrin monomerprecursor of formulae IVa, IVb, IVc, or a mixture thereof, as describedabove. In another preferred embodiment, an oxidized diaminatedcyclodextrin monomer precursor of formula VIIIa, VIIIb, VIIIc or amixture thereof:

may be prepared by amination of an oxidized diiodinated cyclodextrinmonomer precursor of formulae VIIa, VIIb, VIIc, or a mixture thereof, asdescribed above. In still another preferred embodiment, an oxidizeddiaminated cyclodextrin monomer precursor of formula IXa, IXb, IXc or amixture thereof:

may be prepared by displacement of the iodo or other appropriate leavinggroups of an oxidized cyclodextrin monomer precursor disubstituted withan iodo or other appropriate leaving group with the amino or othernucleophilic group containing moiety such as, e.g. HSCH₂CH₂NH₂ (or adi-nucleophilic molecule more generally represented by HW—(CR₁R₂)_(n)—WHwherein W, independently for each occurrence, represents O, S, or NR₁;R₁ and R₂, independently for each occurrence, represent H,(un)substituted alkyl, (un)substituted aryl, (un)substitutedheteroalkyl, (un)substituted heteroaryl) with an appropriate base suchas a metal hydride, alkali or alkaline carbonate, or tertiary amine.

Alternatively, an oxidized diiodinated or diaminated cyclodextrinmonomer precursor, as described above, may be prepared by oxidizing acyclodextrin monomer precursor to form an oxidized cyclodextrin monomerprecursor and then diiodinating and/or diaminating the oxidizedcyclodextrin monomer, as described above. As discussed above, thecyclodextrin moiety may be modified with other leaving groups other thaniodo groups and other amino group containing functionalities. Theoxidized diiodinated or diaminated cyclodextrin monomer precursor maythen be copolymerized with a comonomer A precursor, as described above,to form a linear oxidized cyclodextrin copolymer of the invention.

A linear oxidized cyclodextrin copolymer may also be further modified byattachment of at least one ligand to the copolymer. The ligand is asdescribed above.

According to the invention, a linear cyclodextrin copolymer or linearoxidized cyclodextrin copolymer may be attached to or grafted onto asubstrate. The substrate may be any substrate as recognized by those ofordinary skill in the art. In another preferred embodiment of theinvention, a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer may be crosslinked to a polymer to form,respectively, a crosslinked cyclodextrin copolymer or a crosslinkedoxidized cyclodextrin copolymer. The polymer may be any polymer capableof crosslinking with a linear or linear oxidized cyclodextrin copolymerof the invention (e.g., polyethylene glycol (PEG) polymer, polyethylenepolymer). The polymer may also be the same or different linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer. Thus;for example, a linear cyclodextrin copolymer may be crosslinked to anypolymer including, but not limited to, itself, another linearcyclodextrin copolymer, and a linear oxidized cyclodextrin copolymer. Acrosslinked linear cyclodextrin copolymer of the invention may beprepared by reacting a linear cyclodextrin copolymer with a polymer inthe presence of a crosslinking agent. A crosslinked linear oxidizedcyclodextrin copolymer of the invention may be prepared by reacting alinear oxidized cyclodextrin copolymer with a polymer in the presence ofan appropriate crosslinking agent. The crosslinking agent may be anycrosslinking agent known in the art. Examples of crosslinking agentsinclude dihydrazides and disulfides. In a preferred embodiment, thecrosslinking agent is a labile group such that a crosslinked copolymermay be uncrosslinked if desired.

A linear cyclodextrin copolymer and a linear oxidized cyclodextrincopolymer of the invention may be characterized by any means known inthe art. Such characterization methods or techniques include, but arenot limited to, gel permeation chromatography (GPC), matrix assistedlaser desorption ionization-time of flight mass spectrometry (MALDI-TOFMass spec), ¹H and ¹³C NMR, light scattering and titration.

The invention also provides a cyclodextrin composition containing atleast one linear cyclodextrin copolymer and at least one linear oxidizedcyclodextrin copolymer of the invention as described above. Accordingly,either or both of the linear cyclodextrin copolymer and linear oxidizedcyclodextrin copolymer may be crosslinked to another polymer and/orbound to a ligand as described above. Therapeutic compositions accordingto the invention contain a therapeutic agent and a linear cyclodextrincopolymer or a linear oxidized cyclodextrin copolymer, includingcrosslinked copolymers, of the invention. A linear cyclodextrincopolymer, a linear oxidized cyclodextrin copolymer and theircrosslinked derivatives are as described above. The therapeutic agentmay be any synthetic or naturally occurring biologically activetherapeutic agent including those known in the art. Examples of suitabletherapeutic agents include, but are not limited to, antibiotics,steroids, polynucleotides (e.g., genomic DNA, cDNA, mRNA,double-stranded RNA, and antisense oligonucleotides), plasmids,peptides, peptide fragments, small molecules (e.g., doxorubicin) andother biologically active macromolecules such as, for example, proteinsand enzymes.

(g) Business Methods

Other aspects of the invention provides for certain methods of doingbusiness. In particular, practicing the methods of the invention mayenable novel therapeutic compositions and improved formulations thereof.This technical step, when combined with one or more additional steps,provides for novel approaches to conduct a pharmaceutical, or preferablya life-science business. For example, such therapeutic prepared by themethod of the invention may be tested for efficacy as therapeutics in avariety of disease models, the potential therapeutic compositions thentested for toxicity and other safety-profiling before formulating,packaging and subsequently marketing the resulting formulation for thetreatment of disease. Alternatively, the rights to develop and marketsuch formulations or to conduct such steps may be licensed to a thirdparty for consideration.

Accordingly, in certain embodiments, the present invention provides amethod for conducting a pharmaceutical business, comprising:

a. manufacturing a formulation or kit including a pharmaceuticalcomposition of any of the compounds of claims 1-4; and

b. marketing to healthcare providers the benefits of using theformulation or kit in the treatment of a disease or disorder.

In other embodiments, the present invention discloses a method forconducting a pharmaceutical business, comprising:

a. providing a distribution network for selling a pharmaceuticalcomposition of any of the compounds of claims 1-4; and

b. providing instruction material to patients or physicians for usingthe preparation in the treatment of a disease or disorder.

In certain embodiments, the present invention provides a method forconducting a pharmaceutical business, comprising:

a. determining an appropriate formulation and dosage of a pharmaceuticalcomposition of any of the compounds of claims 1-4;

b. conducting therapeutic profiling of formulations identified in step(a), for efficacy and toxicity in animals; and

c. providing a distribution network for selling a preparation orpreparations identified in step (b) as having an acceptable therapeuticprofile.

An additional step of the embodiment comprises providing a sales groupfor marketing the preparation to healthcare providers.

In still other embodiments, the present invention provides a method forconducting a pharmaceutical business, comprising:

a. determining an appropriate formulation and dosage of a pharmaceuticalcomposition of any of the compounds of claims 1-4; and

b. licensing, to a third party, the rights for further development andsale of the formulation.

EXEMPLIFICATION

Materials. β-Cyclodextrin, “β-CD”, (Cerestar USA, Inc. of Hammond, Ind.)was dried in vacuo (<0.1 mTorr) at 120° C. for 12 h before use.

All the anhydrous solvents, HPLC grade solvents and other common organicsolvents were purchased from commercial suppliers and used withoutfurther purification. Biphenyl-4,4′-disulfonyl chloride (AldrichChemical Company, Inc. of Milwaukee, Wis.) was recrystallized fromchloroform/hexanes. Potassium iodide was powdered with a mortar andpestle and dried in an oven at 200° C. Polyethylene glycoldipropanoicsuccinimide (PEG-DiSPA, MW 3400), polyethylene glycoldibutanoicsuccinimide (PEG-DiSBA, MW 3400), and polyethylene glycoldibenzotrizolecarbonate (PEG-DiBTC, MW 3400) were purchased from Nektar(Huntsville, Ala.). Polyethylene glycol di-p-nitrophenolcarbonate(PEG-DiNPC, MW 3400) was acquired from Sigma (St. Louis, Mo.). CPT waspurchased from Boehringer Ingelheim (Ingelheim, Germany). Human plasmawas purchased from Sigma and reconstituted with DI water. Mouse plasmawas prepared by centrifuge removal of blood cells of fresh blood samplescollected from BALB/C female mice (Charles River).6^(A),6^(D)-diiodo-6^(A),6^(D)-dideoxy-β-cyclodextrin (CDDI, Scheme 2)was synthesized according to previous reported procedure by Hwang et. al(Bioconjugate Chem. 12, 280-290). Deionized water (18-MΩ-cm) wasobtained by passing in-house deionized water through a Barnstead E-purepurification system. NMR spectra were recorded on a Bruker AMX 500 MHzor a Varian 300 MHz spectrometer. Mass spectral (MS) analysis wasperformed using either an electrospray mass spectrometer equipped withLCQ ion trap (Thermo Finnigan) and fitted with an electrosprayionization source or a MALDI-TOF mass spectrometer (Voyager DE-PRO,Applied Biosystems). MWs of the polymer samples were analyzed on a GPCsystem equipped with a Hitachi L-6200 Intelligent Pump, an Anspec RIdetector (ERC-7512, Erma, Inc.), a Precision Detectors DLS detector (PD2020), and double gel permeation columns (PL-aquagel-OH-40 8 μm 300mm×7.5 mm, Polymer Laboratory) calibrated using polyethylene glycolstandard and eluded using PBS (1×) at a concentration of 20-50 mg/mL andat a 0.7 mL/min flow rate at ambient temperature. CD derivatives wereanalyzed with a C-18 reverse phase column on a HPLC system equipped withan UV detector (System Gold 168 Detector, Beckman Coulter) and anevaporative light scattering (ELS) detector (Sedex 75, Sedere, France).CPT, CPT derivatives, and polymer-CPT conjugates were analyzed on HPLCsystems with a C-18 reverse phase column (HIRPB-4438, 4.6×150 mm,Richard Scientific) equipped with a fluorescence detector (FD-500,GTI/Spectro Vision, Groton Technology, Inc.) using a gradient ofpotassium phosphate buffer (pH 4.1) and acetonitile. Excitation andemission wavelengths of the fluorescence detector were set at 370 nm and440 nm, respectively.

Example 1 Biphenyl-4,4′-disulfonyl-A,D-Capped β-Cyclodextrin, 1 (Tabushiet al. J. Am. Chem. Soc. 106, 5267-5270 (1984))

A 500 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 7.92 g (6.98 mmol) of dryβ-cyclodextrin and 250 mL of anhydrous pyridine (Aldrich ChemicalCompany, Inc.). The resulting solution was stirred at 50° C. undernitrogen while 2.204 g (6.28 mmol) of biphenyl-4,4′-disulfonyl chloridewas added in four equal portions at 15 min intervals. After stirring at50° C. for an additional 3 h, the solvent was removed in vacuo and theresidue was subjected to reversed-phase column chromatography using agradient elution of 0-40% acetonitrile in water. Fractions were analyzedby high performance liquid chromatography (HPLC) and the appropriatefractions were combined. After removing the bulk of the acetonitrile ona rotary evaporator, the resulting aqueous suspension was lyophilized todryness. This afforded 3.39 g (38%) of 1 as a colorless solid.

Example 2 6^(A),6^(D)-Diiodo-6^(A),6^(D)-Dideoxy-β-cyclodextrin, 2(Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))

A 40 mL centrifuge tube equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.02 g (7.2 mmol) of 1, 3.54 g(21.3 mmol) of dry, powdered potassium iodide (Aldrich) and 15 mL ofanhydrous N,N-dimethylformamide (DMF) (Aldrich). The resultingsuspension was stirred at 80° C. under nitrogen for 2 h. After coolingto room temperature, the solids were separated by filtration and thesupernatant was collected. The solid precipitate was washed with asecond portion of anhydrous DMF and the supernatants were combined andconcentrated in vacuo. The residue was then dissolved in 14 mL of waterand cooled in an ice bath before 0.75 mL (7.3 mmol) oftetrachloroethylene (Aldrich) was added with rapid stirring. Theprecipitated product was filtered on a medium glass frit and washed witha small portion of acetone before it was dried under vacuum over P₂O₅for 14 h. This afforded 0.90 g (92%) of 2 as a white solid.

Example 36^(A),6^(D)-Bis-(2-aminoethylthio)-6^(A)6^(D)-dideoxy-β-cyclodextrin, 3(Tabushi, I: Shimokawa, K; Fugita, K. Tetrahedron Lett. 1977, 1527-1530)

A 25 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 0.91 mL (7.37 mmol) of a 0.81 M solution of sodium2-aminoethylthiolate in ethanol. (Fieser, L. F.; Fieser, M. Reagents forOrganic Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266). Thesolution was evaporated to dryness and the solid was redissolved in 5 mLof anhydrous DMF (Aldrich).6^(A),6^(D)-Diiodo-6^(A),6^(D)-dideoxy-β-cyclodextrin (2) (100 mg,7.38×10⁻⁵ mol) was added and the resulting suspension was stirred at 60°C. under nitrogen for 2 h. After cooling to room temperature, thesolution was concentrated in vacuo and the residue was redissolved inwater. After acidifying with 0.1 N HCl, the solution was applied to aToyopearl SP-650M ion-exchange column (NH₄ ⁺ form) and the product waseluted with a 0 to 0.4 M ammonium bicarbonate gradient. Appropriatefractions were combined and lyophilized to dryness. This afforded 80 mg(79%) of 3 as a white powder.

Alternative Synthesis of Dicysteamine β-CD 3.

To a solution of 4.69 g (3.17 mmol) of 2 in 100 mL of degassed water wasadded 0.489 g (6.34 mmol) of freshly sublimed cysteamine. The solutionwas stirred under reflux for 2 h. After cooling to room temperature andacidifying with 1 N HCl, the solution was applied to a Toyopearl SP-650Mion-exchange column (NH₄ ⁺ form) and the product was eluted with a 0 to0.2 M ammonium bicarbonate gradient. Appropriate fractions were combinedand lyophilized to dryness. This procedure gave 1.87 g (39% yield) of awhite solid. The solid was characterized by TLC (silica gel,n-PrOH-AcOEt-H₂O—NH_(3aq) 5/3/3/1, detection by ninhydrin) and exhibiteda major spot corresponding to 3. Matrix-assisted laserdesorption/ionization (MALDI) time-of flight (TOF) mass spectrum wasrecorded on 2 meter ELITE instrument supplied by PerSeptive Biosystems,Inc. MALDI-TOF m/z calcd for 3: 1252. found: 1253.5 [M+H]⁺, 1275.5[M+Na]⁺, 1291.4 [M+K]⁺. ¹³C NMR (Bruker 500 MHz, D₂O) δ ppm: 32.1(S—CH₂) and 38.8 (CH₂—NH₂), 32.9 (C6 adjacent to S), 60.2 (C6 adjacentto OH), 70.8, 71.4, 72.5 (C2, C3, C5), 81.8 (C4), 101.7 (C1).

Example 46^(A),6^(D)-Bis-(2-amino-2-carboxylethylthio)-6^(A)′6^(D)-dideoxy-β-cyclodextrin,4 (CD-BisCys)

167 mL of 0.1 M sodium carbonate buffer were degassed for 45 minutes ina 500 mL 2-neck round bottom flask equipped with a magnetic stir bar, acondenser and septum. To this solution were added 1.96 g (16.2 mmol) ofL-cysteine and 10.0 g (73.8 mmol) of diiodo, deoxy-β-cyclodextrin 2. Theresulting suspension was heated at a reflux temperature for 4.5 h untilthe solution turned clear (colorless). The solution was then cooled toroom temperature and acidified to pH 3 using 1N HCl. The product wasprecipitated by slow addition of acetone (3 times weight ratio of thesolution). This afforded 9.0 g crude material containing CD-biscysteine(90.0%), unreacted cyclodextrin, CD-mono-cysteine and cystine. Theresulting solid was subjected to anionic exchange column chromatography(SuperQ650M, Tosoh Bioscience) using a gradient elution of 0-0.4Mammonium bicarbonate. All fractions were analyzed by HPLC. The desiredfractions were combined and the solvent was reduced to 100 mL undervacuum. The final product was either precipitated by adding acetone orby adding methanol (3 times weight ratio of the solution). 4 wasobtained in 60-90% yield. ¹H NMR (D₂O) δ 5.08 (m, 7H, CD-2-CH),3.79-3.94 (m, 30H, CD-3,4-CH, CD-CH₂, Cys-CH), 3.49-3.62 (m, 14H, CD-5,6-CH), 2.92-3.30(m, 4H, Cys-CH₂). ¹³C NMR (D₂O) δ 172.3, 101.9, 83.9,81.6, 81.5, 73.3, 72.2, 72.0, 60.7, 54.0, 34.0, 30.6. ESI/MS (m/z): 1342[M]⁺, 1364 [M+Na]⁺. Purity of 4 was confirmed by HPLC.

Example 56^(A),6^(D)-Bis-(carboxylmethylthio)-6^(A)′6^(D)-dideoxy-β-cyclodextrin,5 (CDDM)

A 50 mL of 0.1 M sodium carbonate solution was degassed for 2 h in a 100mL 3-neck round bottom flask equipped with a magnetic stir bar, acondenser and septa. Mercaptoacetic acid (0.46 mL, 6.64 mmol) wassyringed into the flask and pH of the solution was adjusted to 9.3 with1N sodium hydroxide. To this resulting solution was added 3.00 g (2.21mmol) of di-iodo-β-cyclodextrin 2 and heated at 80° C. for an hour. Thesolution temperature was increased 10° C. every hour until it reached100° C. After 3 h. at the reflux temperature, the clear colorlesssolution was cooled to room temperature and acidified to pH 3.5 using 1N HCl. The crude product was crashed out by slow addition of acetone (3times weight ratio of the solution). The resulting solid was subjectedto anionic exchange column chromatography using a gradient elution of0-0.4 M ammonium bicarbonate solution. This afforded 1.8 g (63.4%) of 5as a colorless solid. ESI/MS (m/z): 1281 [M]⁻. Purity of this compoundwas confirmed with HPLC.

Example 6 CD-Bis(Glutamic acid-γ-Benzyl ester) 6

A 50 mL round bottom flask equipped with a magnetic stirbar and acondenser and a septum was charged with 0.101 g (0.425 mmol) ofH-Glu(Obzl)-OH and 0.15 g (0.106 mmol) of dio-iodo β cyclodextrin 2 in 5mL of degassed 0.1 M sodium carbonate solution. The solution mixture washeated at 100° C. for 2 h. The solution was then cooled to roomtemperature and acidified to pH 4 before dialyzing in MWCO 500 membranesfor 24 h. The yield of 6 was 0.142 g (83.6%).

Example 7 CD-BisLys(Z) 7

A 50 mL round bottom flask equipped with a magnetic stirbar and acondenser and a septum was charged with 0.124 g (0.443 mmol) ofH-Lysine(Z)—OH and 0.15 g (0.111 mmol) of di-iodo-β-cyclodextrin 2 in 5mL of degassed 0.1M sodium carbonate solution. The solution mixture washeated at 100° C. for 4 h. The solution was then filtered and the pH ofthe filtrate is adjusted to 8.5 before dialyzing in MWCO 500 membranesfor 24 h. The yield of 7 was 0.124 g (68.9%).

Example 8 Synthesis of β-cyclodextrin-Tosylate, 8 (Melton, L. D., andSlessor, K. N., Carbohydrate Research, 18, p. 29 (1971))

A 500 mL round-bottom flask equipped with a magnetic stirbar, a vacuumadapter and a septum was charged with a solution of dry β-cyclodextrin(8.530 g, 7.51 mmol) and 200 mL of dry pyridine. The solution was cooledto 0° C. before 1.29 g (6.76 mmol) of tosyl chloride was added. Theresulting solution was allowed to warm to room temperature overnight.The pyridine was removed as much as possible in vacuo. The resultingresidue was then recrystallized twice from 40 mL of hot water to yield7.54 (88%) of a white crystalline solid 8.

Example 9 Synthesis of Iodo-β-cyclodextrin, 9

A round bottom flask with a magnetic stirbar and a Schlenk adapter ischarged with 8, 15 equivalents of potassium iodide, and DMF. Theresulting mixture is heated at 80° C. for 3 h, after which the reactionis allowed to cool to room temperature. The mixture is then filtered toremove the precipitate and the filtrate evaporated to dryness andredissolved in water at 0° C. Tetrachloroethylene is added and theresulting slurry stirred vigorously at 0° C. for 20 minutes. The solid 9is collected on a medium glass frit, triturated with acetone and storedover P₂O₅.

Example 10 Synthesis of Cysteamine-β-Cyclodextrin, 10

To a solution of 9 in 100 mL of degassed water is added 1 eq. of freshlysublimed cysteamine. The solution is stirred under reflux for 2 h. Aftercooled to room temperature and acidified with 1 N HCl, the solution isapplied to a Toyopearl SP-650M ion-exchange column (NH₄ ⁺ form) and theproduct is eluted with an ammonium bicarbonate gradient. Appropriatefractions are combined and lyophilized to dryness to yield 10.

Example 11 Synthesis of Gly-CPT 11 (Greenwald et al., Bioorg. Med.Chem., 1998, 6, 551-562)

t-Boc-glycine (0.9 g, 4.7 mmol) was dissolved in 350 mL of anhydrousmethylene chloride at room temperature, and to this solution were addedDIPC (0.75 mL, 4.7 mmol), DMAP (382 mg, 3.13 mmol) and camptothecin(0.55 g, 1.57 mmol) at 0° C. The reaction mixture was allowed to warm toroom temperature and left for 16 h. The solution was washed with 0.1 NHCl, dried and evaporated under reduced pressure to yield a white solid,which was recrystallized from methanol to give camptothecin-20-ester oft-Boc-glycine: ¹H NMR (DMSO-d₆) 7.5-8.8 (m), 7.3 (s),5.5 (s), 5.3 (s), 4(m), 2.1 (m), 1.6 (s), 1.3 (d), 0.9 (t). Camptothecin-20-ester oft-Boc-glycine (0.595 g, 1.06 mmol) was dissolved in a mixture ofmethylene chloride (7.5 mL) and TFA (7.5 mL) and stirred at roomtemperature for 1 h. Solvent was removed and the residue wasrecrystallized from methylene chloride and ether to give 0.45 g of 11.¹H NMR (DMSO-d₆) δ7.7-8.5 (m); 7.2 (s), 5.6 (s), 5.4 (s), 4.4 (m), 2.2(m), 1.6 (d), 1.0 (t), ¹³C NMR (DMSO-d₆) δ168.6, 166.6, 156.5, 152.2,147.9, 146.2, 144.3, 131.9, 130.6, 129.7, 128.8, 128.6, 128.0, 127.8,119.0, 95.0, 77.6, 66.6, 50.5, 47.9, 30.2, 15.9, 7.9. ESI/MS (m/z)expected 405. Found 406 (M+H).

Example 12 Synthesis of GlyGlyGly-CPT 12

t-Boc-GlyGlyGly (1.359 g, 4.7 mmol) was dissolved in 350 mL of anhydrousmethylene chloride at room temperature and to this solution were addedDIPC (0.75 mL, 4.7 mmol), DMAP (382 mg, 3.13 mmol) and camptothecin(0.55 g, 1.57 mmol) at 0° C. The reaction mixture was allowed to warm toroom temperature and left for 16 h. The solution was washed with 0.1 NHCl, dried and evaporated under reduced pressure to yield a white solid,which was recrystallized from methanol to give camptothecin-20-ester oft-Boc-GlyGlyGly: ¹H NMR (DMSO-d₆) δ 8.40 (s), 8.25 (d), 7.91 (d), 7.78(m), 7.65 (t), 7.26 (s), 7.05 (br, s), 5.65 (d), 5.40 (d), 5.25 (s),5.10 (br, s), 3.75-4.42 (m), 2.15-2.35 (m), 1.45 (s), 0.95 (t)Camptothecin-20-ester of t-Boc-GlyGlyGly (1.5 g, 1.06 mmol) wasdissolved in a mixture of methylene chloride (10 mL) and TFA (10 mL) andstirred at room temperature for 1 h. Solvent was removed under vacuumand the residue was re-dissolved in methylene chloride. The solution waspoured into ether to give instant precipitate (yellow). The precipitatewas filtered and washed with cold ether to give 1.31 g of 12. ¹H NMR(DMSO-d₆) δ 8.79 (s), 7.75-8.61 (m), 7.10 (s), 5.55 (s), 3.90-4.37 (m),3.86 (s), 3.54 (s), 2.11-2.23 (m), 0.95 (t). ESI/MS (m/z) expected 519.Found 520 (M+H).

Stability of CPT-Peptide Ester Bond

11 and 12 were dissolved in PBS buffer (pH 7.4) at room temperature toprepare a solution about 500 μg/mL. This solution was further diluted in8.5% H₃PO₄ to 10 μg/mL. Hydrolysis rate was analyzed using HPLC equippedwith a C₁₈ RP (reverse phase) column and a fluorescence detector using a50/50 (v/v) of acetonitrile/potassium phosphate buffer (pH 4.1). Thepeaks of 11 (or 12) and the released CPT (lactone form) were integrated.The stability of the ester bond in aqueous solution is peptide-lengthdependant. Thus the drug release rate (hydrolysis rate) can be tuned byadjusting the peptide length. See FIG. 2.

Lactone Stability of CPT, 11 and 12 in Phosphate Buffered Saline (PBS)

CPT, 11 or 12 was dissolved in DMSO at 1 mg/mL and then diluted to 1μg/mL with PBS (1×, pH 7.4). 30 μL of solution were injected into theHPLC at room temperature at selected time intervals. The peak area fromthe CPT lactone form of CPT, 11 or 12) were integrated.

The rate of lactone ring opening for 11, 12 and CPT were studied in PBSbuffer (pH 7.4). Both 11 and 12 were very stable against ring-openingand no carboxylate forms of 11 and 12 were detected throughout the study(7 hours). On the other hand, more than 60% of the CPT lactone form wastransformed to its carboxylate form in the same period of time. (SeeFIG. 3)

Example 13 Synthesis of Lys(BisCBZ)-CPT 13

N,N-BisCBZ-Lysine (311 mg, 0.75 mmol) was dissolved in 56 mL ofanhydrous methylene chloride at room temperature. To this solution wereadded DIPC (0.12 mL, 0.75 mmol), DMAP (0.61 mg, 0.5 mmol) andcamptothecin (0.087 g, 0.25 mmol) at 0° C. The reaction mixture wasallowed to warm to room temperature and left for 16 h. The solution waswashed with 0.1 N HCl, dried and evaporated under reduced pressure toyield a light yellow solid, which was recrystallized from methanol togive camptothecin-20-ester of N,N-BisCBZ-Lys 13. Purification of 13 wassatisfactory based on TLC and HPLC analysis.

Hydrolysis of 13 in aqueous solution is very slow and cannot be detectedusing HPLC equipped with a UV detector. Hydrolysis rate of the esterbond of CPT-peptide linker can be tuned not only by adjusting the lengthof peptide, as shown in Example 12, but also by using different aminoacid linked directly with CPT's 20-OH.

The transformation of lactone form to carboxylate form of CPT and 13were also tested in PBS buffer. It was found that the transformation oflactone form to carboxylate form of compound 13 was much slower thanthat of free CPT, indicating that lactone form (drug active form) can bestabilized by forming an ester with the —OH of CPT at its 20 position.(See FIG. 4)

Example 14 Synthesis Lys-Gly-CPT 14

11 is dissolved in chloroform. N,N-DiBoc-Lys-NHS (1.0 eq) is addedfollowed by triethylamine (1.0 eq). The mixture is stirred at rt for 16hours and extracted twice with water and then dried with MgSO4. Solventis removed under high vacuum to yield N,N-DiBoc-Lys-Gly-CPT. To thiscompound is added a mixture of equal volume CH₂Cl₂ and TFA and stirredat rt for 1 h. The solvent is then removed under vacuum. The residue isredissolved in CHCl₃. Ether is added to the solution to crash out theproduct 14. The precipitate is washed several times with ether and thendried under vacuum. It is purified using a silica gel columnchromatography to yield 14 in pure TFA salt form.

Example 15 Synthesis of Suc-Gly-CPT 15

A solution of succinic anhydride is mixed with 11 (1 eq) in dry CHCl₃ inthe presence of a catalytic amount of DMAP and DIEA (1 eq). The mixtureis stirred at rt for 24 hours to yield 15. 15 is purified bycrystallization.

Example 16 Synthesis of Glu-Suc-Gly-CPT 16

15 is converted to its NHS ester using traditional DCC/NHS method. TheNHS ester of 15 is then reacted with glutamic acid (1.0 eq) in DMSO inthe presence of triethylamine. The solution is added to ether toprecipitate 16. 16 is purified by crystallization.

Example 17 Synthesis of Glu-Bis(GlyCPT) 17

11 and Boc-Glu(NHS)—NHS (0.4 eq) are mixed in CHCl₃ under argon beforetriethylamine (1 eq) is added to the mixture. The solution is stirred atrt for 16 h and then washed with acidic water. The organic layer isdried and then solvent is removed under vacuum. The resulting compoundis purified using a silica gel column chromatography. The purifiedcompound is then dissolved in an equal volume mixture of TFA and CH₂Cl₂.The mixture is stirred at rt for 1 h and then poured into ether. Theprecipitate 17 is washed with ether and dried under vacuum.

Example 18 Synthesis of Cyclodextrin-Camptothecin 18

CPT (197 mg, 0.566 mmol) was vacuumed for 30 minutes. Dry chloroform(100 mL) was added under argon. Phosgene (1.34 mL, 20% in toluenesolution) was added at 0° C. Ice bath was removed and the solution waswarmed up to room temperature. Two hours later solvent was removed underhigh vacuum. Dry DMSO (50 mL) was added to the residue, followed by 200mg CD-NH₂ (Cyclodextrin Technology, Inc.) and triethylamine (4 mL,excess). 16 hours later, the solution was poured into 200 mL ether.Precipitate was washed with ether extensively and then dried. 167 mgyellow powder (18) was obtained (62% yield). TLC analysis (silica gel)of 18: R_(f)=0 (developed with CHCl₃/MeOH v/v=5/1). TLC analysis of CPT:R_(f)=0.65 (developed with CHCl₃/MeOH v/v=5/1). Solubility: >10 mg/mL inwater. This indicates that solubility of CPT in water can besubstantially increased when it is covalently attached to cyclodextrinmolecule (free CPT solubility in water <0.004 mg/mL).

Example 19 Synthesis of CDDC-Dianhydride Copolymer 19, 21 and its CPTConjugate 20, 22

A: Ethylenediamine tetraacetic dianhydride (25.6 mg, 0.1 mmol) and CDDC(3, 125.3 mg, 0.1 mmol) were dissolved in 2 mL of dry DMSO. The solutionwas heated at 50° C. for 72 h. Water was added to the mixture, followedby addition of 1 N NaOH to pH around 12. The polymer was dialyzed in10,000 MWCO membrane for 24 h. Precipitation was observed in thedialysis membrane. The solid was removed and the remaining solution wasdialyzed again in 10,000 MWCO membrane for 24 h. A white powder 19 (75mg) was obtained after lyophilization.

11 is added to the polymer (19)/DMSO solution in the presence of EDC (2eq), NHS (1 eq), and DIEA (1.0 eq). The solution is stirred for 16 h andthen poured into ether. The precipitate is washed with CH₂Cl₂extensively until no free drug is observed in the washing solution.Compound 20 is obtained after drying under high vacuum.

B: Diethylenetriamine pentaacetic dianhydride (8.5 mg, 0.024 mmol) andCDDC, 3 (30 mg, 0.024 mmol) were dissolved in 1-methyl-2pyridinone (2mL). The mixture was stirred at 64° C. for 4 days and then dialyzed in10,000 MWCO membrane for 2 days. A white powder 21 (3 mg) was obtainedafter lyophilization.

11 is added to the polymer (21)/DMSO solution in the presence of EDC (2eq), NHS (1 eq), and DIEA (1.0 eq). The solution is stirred for 16 h andthen precipitated in ether. The precipitate is washed with CH₂Cl₂extensively until no free drug is observed in the washing solution.Compound 22 is obtained after drying under high vacuum.

Example 20 Synthesis of CCD-Cys Copolymer 23 and its CPT Conjugate 24

CCD 1 (141.3 mg, 0.1 mmol) and cystine (24 mg, 0.1 mmol) were dissolvedin dry DMSO (0.3 mL) and pyridine (0.1 mL). The mixture was stirredunder argon for overnight at 72° C. Water (10 mL) was added. Precipitatewas filtered and the filtrate was dialyzed in 10,000 MWCO membrane(Spectra/Por 7) for 48 h. A white powder 23 (8 mg) was obtained.

11 is mixed with 23 in DMSO. EDC (2 eq), NHS (1 eq), and DIEA (1.0 eq)are added to the solution. The solution is stirred for 16 h and thenprecipitated with ether. The precipitate is washed with CH₂Cl₂extensively until no free drug is observed in the washing solution.Compound 24 is obtained after drying under high vacuum.

Example 21 Synthesis of CD-BisGlu-Diamine Copolymer 25 and its CPTConjugate 26

CD-Bis(Glutamic acid-γ-Benzyl ester) 6 and ethyleneglycolbisethylamineare dissolved in dry DMSO. EDC (3 eq) and Sulfo-NHS (2 eq) are added tothe mixture. The solution is stirred under argon for 2 days at rt. Thesolution is then transferred to a 10,000 MWCO dialysis membrane anddialyzed for 48 hours. After lyophilization a white powder is obtained.The solid is then dissolved in DMSO and methanol solvent mixture andtreated with H₂ in the presence of 10% Pd/C catalyst for 24 hours. Thesolution is poured into ether to crash out the product. 25 is obtainedafter drying under vacuum.

11 is mixed with 25 in DMSO solution. EDC (2 eq), NHS (1 eq), and DIEA(1.0 eq) are added to the solution. The solution is stirred for 16 hrsand then precipitated with ether. The precipitate is washed with CH₂Cl₂extensively until no free drug is observed in the washing solution.Compound 26 is obtained after drying under high vacuum.

Example 22 Synthesis of CDDM-Lys(GlyCPT) Polymer 27

CDDM, 5, and Lys-Gly-CPT, 14, are dissolved in dry DMSO. EDC (3 eq) andSulfo-NHS (2 eq) are added to the mixture. The solution was stirredunder argon for 2 days at rt. The solution is then poured into ether.The precipitate 27 is dried under vacuum.

Example 23 Synthesis of CDDC-Cys(Boc) Copolymer 28, CDDC-CysCopolymer 29and its CPT Conjugate 30

CDDC (3) and N,N-DiBoc-Cystine are dissolved in dry DMSO. EDC (3 eq) andSulfo-NHS (2 eq) were added to the mixture. The solution was stirredunder argon for 2 days at rt. The solution is then transferred to a10,000 MWCO dialysis membrane and dialyzed for 48 hours. Afterlyophilization a white powder, CDDC-Cys(Boc) polymer 28, is obtained. Tothe white powder 28 is added a mixture of HCl and DMSO solution. Thesolution is stirred at rt for 1 h and then dialyzed against water for 24h using 10,000 MWCO membrane. 29 is obtained as a white solid.

Suc-Gly-CPT 15 is mixed with 29 in DMSO solution. EDC (2 eq), NHS (1eq), and DIEA (1.0 eq) are added to the solution. The solution isstirred for 16 h under argon and then precipitated with ether. Theprecipitate is washed with ether until no free drug is observed in thewashing solution. Compound 30 is obtained after drying under highvacuum.

Example 24 Synthesis of Biodegradable CD-Polyphosphoester Polymer 31 andits CPT Conjugates 32

Synthesis of the biodegradable polyphosphoester can be found in Wang Jet al, JACS, 2001, 123, 9480-9481.

Polyphosphoester is mixed with 10 (0.5 eq of repeat unit) in DMSO. EDC(2 eq), NHS (1 eq), and DIEA (1.0 eq) are added to the solution. Thesolution is stirred for 16 hrs and then precipitated with ether. Theobtained CD-polyphosphoester 31 is dissolved in DMSO. To the solution isadded 11 (0.5 eq of repeat unit), EDC (2 eq), NHS (1 eq), and DIEA (1.0eq). The solution is stirred for 16 h and then precipitated with ether.The precipitate is washed with ether extensively until no free drug isobserved in the washing solution. Compound 32 is obtained after dryingunder high vacuum.

Example 25 Synthesis of CD Copolymer—CPT Conjugate 33 with PolyethyleneBackbone Via Radical Polymerization

Acrylate monomers of CPT, triethyleneglycol monomethylether, andCD-monocystamine can be synthesized from N-Acryloxysuccinimide(Polysciences, Inc.). These monomers are mixed in 1:1:1 ratio in dryDMSO. AIBN is added to the mixture under argon. The solution is stirredat rt for 24-48 hrs until the solution becomes viscous. Polymer-CPTconjugate 33 is precipitated with ether and dried under vacuum.

Example 26 Synthesis of CD-graft-poly(ethylene-alt-maleicanhydride)-GlyGlyGlyCPT 34

Poly(ethylene-alt-maleic anhydride) (Aldrich) is dissolved in DMSO. 10(0.4 eq of repeat unit) and 12 (0.4 eq of repeat) are added. Thesolution is heated at 70° C. for 16 hrs and then precipitated withether. The obtained CD-graft-poly(ethylene-alt-maleicanhydride)-GlyGlyGlyCPT 34 is dried under high vacuum.

Example 27 Synthesis of Polyglutamate-CD-CPT Conjugate 35

Polyglutamate (from Sigma-Aldrich) is mixed with 10 (0.5 eq of repeatunit) and 11 (0.5 eq of repeat unit) in DMSO. EDC (3 eq), NHS (2 eq),and DIEA (1.0 eq) are added to the solution. The solution is stirred for16 hr and then precipitated with ether. After drying under high vacuum,polyglutamate-CD-CPT conjugate 35 is obtained.

Example 28 Synthesis and Characterization of CD-BisCys-Peg3400Copolymers 36 and their CPT Conjugates 37 A. Synthesis andCharacterization of CD-BisCys-Peg3400 Copolymers 36

Synthesis of Poly(CDDCys-PA-PEG), 36a 4 (after precipitation withacetone, 63 mg, 0.047 mmol) and PEG-DiSPA (MW 3400, 160 mg, 0.047 mmol)were dried under vacuum for 8 hours. Anhydrous DMSO (1.26 mL) was addedto the mixture under argon. After 10 minutes of stirring, anhydrousdiisopropylethylamine (DIEA, 19 μL, 2.3 eq.) was added under argon. Thereaction mixture was stirred under argon for 120 h. The polymercontaining solution was dialyzed using a 10,000 MWCO membrane(Spectra/Por 7) against water for 48 h and lyophilized to yield 196 mg36a (90%, Table 1). M_(w)=57.4 kDa, M_(n)=41.7 kDa, M_(w)/M_(n)=1.38. ¹HNMR (D₂O) δ 5.08 (m, CD-2-H), 4.27 (m, Cys-CH), 2.72-3.76 (m,CD-3,4,5,6-CH, CD-CH₂, PEG-CH₂), 2.44 (m, Cys-CH₂).

Synthesis of other poly(CDDCys-PA-PEG) (36b-f), Poly(CDDCys-BA-PEG)(36g) Poly(CDDCys-CB-PEG) (36h-i) were achieved under polymerizationcondition similar to that of 36a. Details for the polymerizationconditions, monomer selection, polymer molecular weight, polydispersityand yields are listed in Table 1. 36g: ¹H NMR (D₂O) δ 5.10 (m, CD-2-H),4.25-4.37 (m, Cys-CH), 2.72-3.86 (m, CD-3,4,5,6-CH, CD-CH₂, PEG-CH₂),2.21 (m, Cys-CH₂). 36h-i: ¹H NMR (D₂O) δ 5.05 (m, CD-2-H), 4.56 (m,Cys-CH), 2.70-3.93 (m, CD-3,4,5,6-CH, CD-CH₂, PEG-CH₂), 2.38 (m,—OCH₂CH₂CH₂C(O)—NH—), 2.34 (m, Cys-CH₂), 1.90 (m, —OCH₂CH₂CH₂C(O)—NH—).

Addition of a non-nucleophilic organic base (such as DIEA) was essentialfor this polymerization as no viscosity changes of the polymerizationsolutions were observed after 48 hours if no base was added. When 2.3eq. of DIEA were added, the viscosity of the polymerization solutionincreased dramatically after 4-6 hours of reaction. DIEA deprotonatesthe amino groups of 4 to render them more nucleophilic for coupling withPEG-DiSPA. There were essentially no differences in the polymerizationsif other bases, such as TEA or DMAP, were used (36b-c, Table 1).Polymerization using 4 recovered by the two different precipitationmethods (acetone and methanol) produced polymers with different MWs. 4that was purified by the methanol-precipitation method (contains no freecystine) gave higher MW polymer (36d-e) as compared to the less pure 4that was obtained from the acetone-precipitation method (36a).Polymerization of 4 with PEG-DiSPA typically produced polymer yieldsgreater than 90%.

4 was polymerized with other activated monomers such as PEG-DiSBA,PEG-DiBTC, and PEG-DiNPC. Reaction of 4 with PEG-DiSBA gave polymer 36gwith similar linkages as 36a-f (amide bond, but one more —CH₂ group than36a-f at the linker) with M_(w) over 100 kDa, while reaction of 4 withPEG-DiBTC and PEG-DiNPC generated polymers 36h and 36i, respectively,with connecting carbamate moiety and M_(w)'s over 50 kDa (Table 1).

TABLE 1 Polymerization of 4 with difunctionalized PEG Poly- PEGmerization M_(w) M_(n) M_(w)/ Yield CDP Comonomer Base time (h) (kDa)(kDa) M_(n) (%) 36a^(a) PEG-DiSPA DIEA 120 57.4 41.7 1.38 90 36b^(a)PEG-DiSPA DMAP 120 54.2 38.1 1.42 91 36c^(a) PEG-DiSPA TEA 120 57.4 42.61.35 91 36d^(b) PEG-DiSPA DIEA 120 93.6 58.0 1.48 96 36e^(b) PEG-DiSPADIEA 144 97.3 58.0 1.67 94 36f^(b) PEG-DiSPA DIEA 2 35.3 25.6 1.38 9536g PEG-DiSBA DIEA 120 114.7 77.9 1.47 96 36h PEG-DiBTC DIEA 120 67.639.4 1.47 95 36i PEG-DiNPC DIEA 120 86.5 57.2 1.51 96 ^(a)4 was washedwith acetone before polymerization. ^(b)4 was washed with methanolbefore polymerization.

Polymers 36a-i are highly soluble in aqueous solution. They can beeasily dissolved in water or phosphate buffered saline (PBS) solution atconcentrations of at least 200 mg/mL. Solubility of these polymers inaqueous solution at concentrations higher than 200 mg/mL was notattempted due to the high viscosity. These polymers were also soluble inDMF, DMSO and methanol, slightly soluble in CH₃CN and CHCl₃, butinsoluble in THF and ethyl ether.

Molecular Weight Control of CD Polymers 4 (after precipitation withmethanol) (56.2 mg, 0.0419 mmol) and PEG-DiSPA (147 mg, 0.0419 mmol)were dried under vacuum for 4-8 hours. To the mixture was added dry DMSO(1.1 mL) under argon. After 10 minutes stirring, DIEA (16 μL, 2.2 eq)was added under argon. A portion of polymerization solution (150 μL) wasremoved and precipitated with ether at selected times (2 h, 18 h, 43 h,70 h, 168 h and 288 h). MWs of the precipitated polymers were determinedas described above.

As shown in FIGS. 5 a and 5 b, molecular weights of 36 can be controlledby adjusting polymerization time.

B. Synthesis of Poly(CDDCys-PA-PEG)-CPTConjugates (HGGG6, LGGG10, HG6,HGGG10)

Synthesis of Poly(CDDCys-PA-PEG)-GlyGlyGly-CPT (HGGG6) 36e (1.37 g, 0.30mmol of repeat unit) was dissolved in dry DMSO (136 mL). The mixture wasstirred for 10 minutes. 12 (419 mg, 0.712 mmol, 2.36 eq), DIEA (0.092mL, 0.712 mmol, 2.36 eq), EDC (172 mg, 0.903 mmol, 3 eq), and NHS (76mg, 0.662 mmol, 2.2 eq) were added to the polymer solution and stirredfor ca. 15 hours. The polymer was precipitated with ethyl ether (1 L).The ether was poured out and the precipitate was washed with CH₃CN(3×100 mL). The precipitate was dissolved in water 600 mL. Someinsoluble solid was filtered through 0.2 μm filters. The solution wasdialyzed using 25,000 MWCO membrane (Spectra/Por 7) for 10 h at 10-15°C. in DI water. Dialysis water was changed every 60 minutes. Thepolymer-drug conjugate solution was sterilized by passing it through 0.2μM filters. The solution was lyophilized to yield a yellow solid HGGG6(1.42 g, 85% yield).

Synthesis of Poly(CDDCys-PA-PEG)-GlyGlyGly-CPT (LGGG10) Conjugation of12 to 36f was performed in a manner similar to that used to produceHGGG6 except that this conjugate was dialyzed with 10,000 MWCO membrane(Spectra/Por 7) instead of with 25,000 MWCO membrane. The yield ofLGGG10 was 83%.

Synthesis of Poly(CDDCys-PA-PEG)-Gly-CPT (HG6) Conjugation of 11 to 36ewas performed in a manner similar to that used to produce HGGG6. Theyield of HG6 was 83%.

Synthesis of Poly(CDDCys-PA-PEG)-GlyGlyGly-CPT (HGGG10) 36e (1.5 g, 0.33mmol of repeat unit) was dissolved in dry DMSO (150 mL). The mixture wasstirred for 10 minutes. 12 (941 mg, 1.49 mmol, 4.5 eq), DIEA (0.258 mL,1.49 mmol, 4.5 eq), EDC (283 mg, 1.49 mmol, 4.5 eq), and NHS (113 mg,0.99 mmol, 3 eq) was added to the polymer solution and stirred for ca.24 hours. Another portion of EDC (142 mg, 0.75 mmol, 2.3 eq) and NHS (56mg, 0.5 mmol, 1.5 eq) were added to the conjugation solution. Thepolymer was stirred for an additional 22 hours. The workup procedure wasthe same as that for the synthesis of HGGG6. The yield of HGGG10 was77%.

Determination of wt % CPT on the Conjugates Stock solutions of HGGG6,LGGG10, HG6 and HGGG10 were prepared at a concentration of 10 mg/mL inDMSO. An aliquot of corresponding stock solution was diluted to 100μg/mL using 1 N NaOH. CPT was completely hydrolyzed in this basicsolution and transformed to its carboxylate form within 2 h at roomtemperature. An aliquot of this solution was diluted to 10 μg/mL using8.5% H₃PO₄, and the CPT carboxylate form was transformed to its lactoneform. 30 μL of this solution was injected into the HPLC. The peak areafrom the CPT lactone form was integrated and compared to a standardcurve.

11 and 12 were conjugated to 36e or 36f (Table 2) using conventionalcoupling methods. Due to the instability of the ester linker of 11 and12 in aqueous solution, the conjugation was conducted in anhydrous DMSOunder argon. An organic base was required to deprotonate the TFA saltsof 11 and 12 to facilitate the coupling. For polymer conjugation with12, the weight percent (wt %) drug loading was around 6-10%. Thetheoretical maximum drug loading is around 13% using PEG with MW of 3400Da; maximum values can be increased by decreasing the MW of the PEGsegments. Solubilities of all conjugates in water or PBS were more than200 mg/mL (equivalent to a 12-20 mg CPT/mL for 6-10 wt % drug loading,respectively). Details for the HGGG6, LGGG10, HG6, and HGGG10 aresummarized in Table 2.

TABLE 2 Properties of polymer-CPT conjugates. M_(w) of parent polymerCPT Conjugate^(a) (×10⁻³) M_(w)/M_(n) ^(b) Linker (wt %) HGGG6 97 1.7triglycine 6.1 LGGG10 35 1.6 triglycine 10.2 HG6 97 1.7 glycine 6.8HGGG10 97 1.7 triglycine 9.6 ^(a)Abbreviations: H = High M_(w) polymer(97 kDa), L = Low M_(w) polymer (35 kDa), GGG = triglycine linker, G =glycine linker, 6 = drug loading around 6 wt %, 10 = drug loading around10 wt %. ^(b)Polymer polydispersity as measured by light scatteringtechniques(26)

C. Release of CPT from HGGG6 and HG6

Release of CPT in PBS HGGG6 and HG6 were prepared at 1 mg/mL in PBS (1×,pH 7.4). A 100 μL aliquot of the solution was transferred to a 1.5 mLEppendorf tube and incubated at 37° C. The incubated samples werequenched at selected time intervals and stored at −80° C. until theanalysis. Each solution was diluted with 8.5% H₃PO₄ to a 5 mL totalvolume in a volumetric flask. 30 μL of such solution was injected intothe HPLC. The peak area from the CPT lactone form was integrated andcompared to a standard curve.

Analysis for the release of CPT from HGGG6 and HG6 in PBS containingacetyl cholinesterase (an esterase, 100 units/mL), in KH₂PO₄ buffer (pH6.1, 0.1 M) and in the KH₂PO₄ buffer (pH 6.1, 0.1 M) containingcathepsin B (a cysteine proteinase, 200 μM, preactivated on ice for 30minutes in this buffer containing 2 mM DTT and 1 mM EDTA) were performedin a manner similar to that described above for PBS alone.

Release of CPT in Human Plasma An aliquot of HGGG6 and HG6 stocksolution were diluted to give final concentration of 0.5 mg/mL in PBS(1×, pH 7.4). This solution was added to a lyophilized powder of humanplasma to reconstitute 100% human plasma by the recommended amount. Thesolution was divided into equal volume (250 μL) to 1.5 mL Eppendorftubes, incubated at 37° C., and stopped at selected time point. Sampleswere stored at −80° C. until the analysis. Samples were separated fromplasma by solid phase extraction columns. The solid phase extractioncartridge (Oasis HLB 1cc cartridge from Waters) was pre-conditioned with1 mL of acetonitrile and then with 1 mL of 8.5% H₃PO₄ before loading.Samples were acidified with equal volume of 8.5% H₃PO₄ prior to loading.After the acidified solution was loaded on the cartridge, the bed waswashed with 3×1 mL of water. Released CPT and polymer conjugate wereeluted with 3×1 mL of a solution mixture of acetonitrile and potassiumphosphate buffer (pH 4.1) (60/40 v/v). The eluted solution was dilutedto 5 mL total volume in a 5 mL volumetric flask. 30 μL of such solutionwas injected into the HPLC. The peak area from the CPT lactone form wasintegrated and compared to a standard curve.

Release of CPT from HGGG6 and HG6 in PBS containing 4% human plasma(PBS/reconstituted human plasma solution=96/4 (v/v)), in mouse plasmaand in reconstituted human albumin (PBS solution) were performed in amanner similar to that described above for pure human plasma.

In PBS (1×, pH 7.4), the half-lives (t_(1/2)) for releasing CPT from HG6and HGGG6 were 59 h and 32 h, respectively. The half-lives decreased to25 h and 22 h, respectively, in the presence of 4% human plasma, and to1.7 h and 1.6 h, respectively, in 100% human plasma (“HP”) and 2.6 h and2.2 h, respectively, in 100% mouse plasma (“MP”). CPT release rates forboth HG6 and HGGG6 in the presence of albumin (“Alb”) or acetylcholinesterase (“Ac Cho”) were on the same order of magnitude as in PBS.In a buffer solution at a pH lower than PBS (pH 6.1) with or without theenzyme cathepsin B (active at pH 6.1), less than 50% of total conjugatedCPT was released from both HG6 and HGGG6 for times up to 144 h (Table3).

TABLE 3 Half-life (t_(1/2), in hour) of the release of CPT from HG6 andHGGG6^(a) 4% Ac pH 6.1 Cath B Conjugate PBS^(b) HP^(c) HP^(d) MP^(e)Alb^(f) Cho^(g) buffer^(h) (pH 6.1)^(i) HG6 59 25 1.7 2.6 6233 >144 >144 HGGG6 32 22 1.6 2.2 73 43 >144 >144 ^(a)t_(1/2) is definedas time (hours) for the release of half of the total conjugated CPT.Abbreviations: HP means human plasma, MP means mouse plasma. ^(b)pH 7.4PBS 1x buffer. ^(c)Reconstituted human plasma mixed with PBS (v/v =4/96). ^(d)Reconstituted human plasma ^(e)Fresh mouse plasma ^(f)Inreconstituted human albumin PBS buffer ^(g)In the presence of acetylcholinesterase PBS solution (100 units/mL). ^(h)pH 6.1 phosphate buffer(0.1M) ^(i)pH 6.1 phosphate buffer in the presence of Cathepsin BRelease of CPT in Solution at Different pH. HGGG6 and HG6 were preparedat 1 mg/mL in buffer solution with pHs ranging from acidic (pH=1.2) tobasic (pH=13.1) and incubated at 37° C. for 24 h. An aliquot of eachsolution was diluted with 8.5% H₃PO₄ to about 100 μg/mL. 30 μL of suchsolution was injected into HPLC. The peak area from the CPT lactone formwas integrated and compared to a standard curve.

The pH of aqueous solution has a significant effect on the CPT releaserates from both HG6 and HGGG6. The amounts of CPT released from HG6 andHGGG6 at 37° C. after 24 h in buffer solutions with pHs ranging from 1.1to 13.1 are illustrated in FIG. 6. The glycinyl-CPT ester bonds of bothHG6 and HGGG6 were very stable in acidic pH (1.1 to 6.4) as less than 7%of CPT were released in 24 h.

IC₅₀ via MTT Assay The human ovarian carcinoma A2780 cell line wasobtained from the European Collection of Cell Cultures (Salisbury,Wiltshire, UK). The human colorectal adenocarcinoma HT29, human prostatecarcinoma PC-3, and human colonic adeoncarcinoma LS 174T cell lines wereobtained from the American Type Culture Collection (Rockville, Md.).Cells were seeded in 96-well plates at 5000 cells/well and grown inmedium containing 10% fetal bovine serum at 37° C. for 24 h in ahumidified 5% CO₂ atmosphere. The medium was replaced with fresh mediumcontaining CPT, 36e, HGGG6 or HG6 in concentrations ranging from 1 nM to10 μM of CPT and 36e (CPT equivalent for HGGG6 and HG6). At eachconcentration three wells per plate were treated. The effect of thecompounds on cell growth was measured by the MTT assay after 72 h. Themedium was removed, the cells were rinsed with PBS, MTT solution wasadded at a concentration of 0.5 mg/mL, and the plates were incubated for4 h at 37° C. The medium was removed and the formazan crystals weresolubilized in DMSO. Absorbance was measured at 560 nm using aSPECTRAFluor Plus plate reader (Tecan, Durham, N.C.). The percentage ofcell survival was calculated relative to untreated cells, and IC₅₀'swere determined from plots of dose versus cell survival. IC50 data ofCPT, 36e, HGGG6 or HG6 are listed in Table 4.

TABLE 4 IC₅₀ of CPT, unconjugated polymer 36e and CPT conjugates HG6 andHGGG6 in various cell lines Cell Line 36e (μM) CPT (μM) HG6 (μM) HGGG6(μM) LS174T >300 0.005 0.050 0.010 HT29 300 0.020 0.050 0.030 A2780 1000.007 0.025 0.020 PC3 >300 0.075 0.25 0.15

Example 29 Poly-CD-BisCys-Peg3400-Ala-CPT 37

36e (54 mg, 0.012 mmol of repeat unit) was dissolved in dry DMSO (226mL) and stirred for 10 minutes. TFA-Ala-CPT which is prepared similar to11 (15 mg, 0.028 mmol, 2.36 eq), DIEA (4.88 mL, 0.028 mmol, 2.36 eq),DCC (24.52 mg, 0.12 mmol, 10 eq), and NHS (13.6 mg, 0.12 mmol, 10 eq)were added to the polymer solution. The mixture was stirred for about 16hours. The polymer was precipitated with ether (40 mL) and washed withether (2×30 mL) and with CH₃CN (2×10 mL). It was then redissolved in pH4 aqueous solution (10 mL) and dialyzed at room temperature for 48 husing 25,000 MWCO membrane. The solution was then passed through asterilized 0.2 μm filter and then lyophilized to yield 37(46 mg, 85%).Weight percent of drug loading was calculated to be 5.5% using HPLCequipped with a fluorescence detector after releasing CPT from 37 usingbase. Free CPT in 37 is <1%.

Example 30 Poly-CD-BisCys-Peg3400-Leu-CPT 38

36e (54 mg, 0.012 mmol of repeat unit) was dissolved in dry DMSO (226mL) and stirred for 10 minutes. TFA-Leu-CPT which is prepared similar to11 (16 mg, 0.028 mmol, 2.36 eq), DIEA (4.88 mL, 0.028 mmol, 2.36 eq),DCC (24.52 mg, 0.12 mmol, 10 eq), and NHS (13.6 mg, 0.12 mmol, 10 eq)were added to the polymer solution. The mixture was stirred for about 16hours. The polymer was precipitated with ether (40 mL) and washed withether (2×30 mL) and with CH₃CN (2×10 mL). It was then redissolved in pH4 aqueous solution (10 mL) and dialyzed at room temperature for 48 husing 25,000 MWCO membrane. The solution was then passed through asterilized 0.2 μm filter and then lyophilized to yield 38 (42 mg, 78%).Weight percent of drug loading was calculated to be 5.0% using HPLCequipped with a fluorescence detector after releasing CPT from 38 usingbase. Free CPT in 38 is <1%.

Example 31 Synthesis of CD-BisCys-BisPeg-FITC 39

4 (25 mg, 0.0186 mmol) and FITC-Peg5000-NHS (Shearwater, 186 mg, 0.0373mmol) were dissolved in dry DMSO (2 mL). DIEA (0.0094 mL, 0.056 mmol, 3eq) was added to the mixture. The mixture was kept in dark and stirredfor 24 hours. Water (10 mL) was then added and the solution was dialyzedin dark using 10,000 MWCO for about 48 hours. After lyophilization ayellow polymer 39 was obtained. Polymer was characterized by MS and ¹HNMR.

Example 32 Synthesis of Bis-succinimidyl succinatePeg3400 (Bis-SS-PEG)(40a) and Biodegradable CD-BisCys-SS-Peg3400 (40b) and its CPT Conjugate41

A 100 mL round bottom flask equipped with a magnetic stirbar and aseptum was charged with 10 g (2.99 mmol) of polyethylene glycol Mw 3350,2.0 g (20 mmol) of succinic anhydride and 50 mL of anhydrous pyridine.After stirring the solution at 50° C. for 16 h, the solvent was removedby rotary evaporator. The residue was redissolved in 30 mL of water andextracted with 10 mL of chloroform three times. The organic layer wasdried over MgSO₄ and filtered. The solvent was then concentrated andprecipitated out in diethyl ether. This resulted in 9.6 g ofbis-succinimidyl Peg3400 at a yield of 90.6%. The product was analyzedby reverse-phase columned High Performance Liquid Chromatography.

A 100 mL round bottom flask equipped with a magnetic stirbar and aseptum was charged with 2 g (0.56 mmol) of bis-succinimidyl Peg3400 and10 mL of anhydrous dichloromethane. To this solution was added 0.324 g(2.82 mmol) of N-hydroxyl succinimide. The solution mixture was thencooled in an ice bath and added 0.58 g (2.82 mmol) of1,3-dicyclohexylcarbodiimide. After leaving at room temperature for 24h, the solution was filtered and precipitated out in 150 mL of diethylether. Dissolution in 10 mL dichloromethane and precipitation in diethylether was repeated two times. This afforded 1.74 g (82.9%) of Bis-SS-PEG40a. It was analyzed by reverse-phase columned High Performance LiquidChromatography.

CD-BisCys-SS-Peg3400 Polymer 40b

A 50-mL pearl shaped flask was charged with 100 mg (0.0746 mmol) of 4and 254 mg (0.0746 mmol) of 40a. The combined solids were dried undervacuum for 24 hours before the addition of 1 mL of anhydrous DMSO and2.2 equivalents (0.164 mmol) of DIEA. The solution mixture was stirredat room temperature for 3 days and then precipitated out in diethylether. This yielded 100% of 40b. Molecular weight was analyzed on aHitachi HPLC system equipped with an Anspec RI detector, a PrecisionDetectors DLS detector, and a Progel-TSK G3000_(PWXL) column using 0.1 MPBS as eluant at a 0.7 mL min⁻¹ flow rate. M_(w)=93,000, M_(n)=61,000and M_(w)/M_(n)=1.5.

CD-BisCys-SS-Peg3400-GlyGlyGly-CPT Conjugate 41

40b (201.8 mg, 0.044 mmol of repeat unit), TFA-GlyGlyGly-CPT 12 (66 mg,0.105 mmol, 2.36 eq), EDC (25.5 mg, 0.133 mmol, 3 eq), and NHS (11 mg,0.0977 mmol, 2.2 eq) were dissolved in dry DMSO (6 mL) and stirred for30 minutes. DIEA (19 μL, 0.105 mmol, 2.36 eq), added to the polymersolution. The mixture was stirred for about 41 hours. The polymer wascrashed out with diethyl ether (250 mL) and washed with acetonitrile(3×25 mL). It was then re-dissolved in pH 4 water (10 mg/mL) anddialyzed at room temperature for 24 hours using 10,000 MWCO membrane.The solution was then passed through a sterilized 0.2 μm filter and thenlyophilized to yield 41 (128 mg, 52%). Weight percent of drug loadingwas calculated to be 6.95% using HPLC equipped with a fluorescencedetector after releasing CPT from 41 using base.

Hydrolysis of 41 was set up in human plasma (100% solution) at 1 mg/mL.Aliquot solutions (100 μL) were placed in 1.5 mL eppendorf tubes andincubated in 37° C. water bath. Then, the samples were acidified with100 μL of 8.5% H₃PO₄ and loaded on pre-conditioned solid phaseextraction cartridge. It was eluted with 60:40 (v/v) acetonitrile:KH₂PO₄ buffer. Free CPT (lactone form) was analyzed on HPLC/Fluorescencedetector using acetonitrile/KH₂PO₄ buffer. Half-life was determined tobe 3 h.

Degradation of CD-BisCys-SS-Peg3400 Polymer 40b

50 mg/mL of 40b solution was prepared in human plasma reconstituted inPBS (pH 7.4) solution. 100 μL aliquots were incubated at 37° C. Eachsample tube was taken out at a specific time point and crashed out in900 μL cold methanol. The solution was centrifuged and the supernatantwas analyzed on a HPLC/ELS detector. The resulting spectrum is shown inFIG. 7.

Methods for Increasing Drug Weight Percent Loading

Method I. Synthesis of CD-BisCys-Peg Copolymer with a Short Peg Linkageand its GlyCPT Conjugate Example 33 Synthesis of CD-BisCys-Peg (ShortPEG, e.g., Peg200-Peg2000) and its CPT Conjugate 42

Synthesis of polymer and drug conjugate 42 are same as 36, 37, and 38 inExample 28.

Method II. Synthesis of CD-BisCys-Peg Copolymer with Multiple DrugMolecules on Each Loading Site Example 34 Synthesis of CD-BisCys-Peg andits GluBis(GlyCPT) Conjugate 43

36 and Glu-Bis(Gly-CPT) 17 are dissolved in DMSO. EDC (3 eq), NHS (2.2eq), and DIEA (2.2 eq) are added to the solution.CD-BisCys-Peg-GluBis(GlyCPT) 43 is precipitated with CH₃CN and washedwith the same solvent until no free drug is detected using UV or TLC. 43is dried under high vacuum.

Example 35 Synthesis of PEI-CD-CPT Conjugate 44 (Branched CD-Polymerwith CPT Conjugates)

A: Synthesis of Branched PEI-Cyclodextrin Polymer

PEI (29 mg, Aldrich Mw 25,000) was dissolved in dry DMSO (2 mL).Cyclodextrin monotosylate (448 mg, Cyclodextrin TechnologiesDevelopment, Inc.) was added to the solution under N₂. The cloudysolution turned clear after the mixture was stirred at 70° C. for about1 hour. The solution turned slightly yellow after 48 hours at suchtemperature under N₂.

The solution was transferred to a Spectra/Por MWCO 10,000 membrane anddialyzed against water for 4 days. Water was then removed bylyophilization. A white powder was obtained (120-140 mg) after thesolution was lyophilized. Cyclodextrin/PEI ratio was calculated based onthe proton integration of ¹H NMR.

B: Synthesis of Branched PEI-CD-CPT Conjugate

PEI-CD and Suc-Gly-CPT 15 (1.0 eq) are dissolved in DMSO. EDC (3 eq),NHS (2.2 eq), and DIEA (1 eq) are added to the solution. PEI-CD-Gly-CPT44 is precipitated with ether, washed extensively with this solvent, anddried under high vacuum.

Example 36 Synthesis of Ad-PEG₃₄₀₀-Ad 45

240 mg of 1-aminoadamantane (1.60 mmol, Aldrich) and 272 mg ofPEG₃₄₀₀(SPA)₂ (0.080 mmol, Shearwater Polymers) was added to a glassvial equipped with a stirbar. To this was added 5 mL of dichloromethane,and the solution was stirred overnight. The next day, the solution wasfiltered to remove the n-hydroxysuccinimide byproduct and thedichloromethane was removed in vacuo. The residue was dissolved in waterand centrifuged to remove excess 1-aminoadamantane. The supernatant wasthen dialyzed overnight in Pierce's Slide-A-Lyzer with MWCO=3500. Thesolution was then lyophilized to afford 248 mg of a white fluffy solidof Ad-PEG₃₄₀₀-Ad 45.

Example 37 Synthesis of DiCyclodextrin PEG 46

362 mg of CD-NH₂ (0.32 mmol, Cyclodextrin Technology, Inc.) and 436 mgof PEG₃₄₀₀(SPA)₂ (0.128 mmol, Shearwater Polymers) were added to a glassvial equipped with a stirbar. To this vial was added 4.36 mL of DMSO,and the solution was stirred for 72 hrs. The solution was dialyzed using2000 MWCO membrane for 4 days in water. 46 (603 mg, 86%) was obtained asa white powder after lyophilization.

Example 38 Synthesis of Inclusion Polymer 47 Using DiAD-Peg 45 andDiCD-PEG 46

46 (54.6 mg, 0.01 mmol) and 45 (34 mg, 0.01 mmol) were mixed in water(0.27 mL) and stirred for overnight. The solution is very viscous.Polymer 47 was crashed out with ether and dried under vacuum

Example 39 Synthesis of Inclusion Polymer CPT-Conjugate 48 BetweenDiCD-PEG 46 and a CPT-Di-AD Compound Synthesis of DiadamantaneCrosslinker: Bis-(2(1-Adamantyl)Ethyl)Phosphate

(Zhang et al., J. Am. Chem. Soc, 1997, 119, 1676-1681)

Anhydrous pyridine (10 mL, Aldrich, Milwaukee, Wis.) was cooled in anice bath and methyl dichlorophosphate (1.488 g, 10 mmol, Aldrich,Milwaukee, Wis.) was added dropwise. The mixture was kept cold for afurther 15 min. During this period a precipitate of N-methylpyridiniumdichlorophosphate formed. 1-Adamantane ethanol (4.758 g, 26.4 mmol,Aldrich, Milwaukee, Wis.) was added, and the sealed mixture was stirredovernight at room temperature. It was then poured into 10% NaHCO₃solution (50 mL) and the pyridine was evaporated under vacuum. Theslightly yellow solid was dissolved in 1 L of water and extracted withether (three 150 mL portions). The aqueous phase was acidified with 2 NHCl to pH 1, and then extracted with three 150 mL portions ofCHCl₃:n-BuOH (7:3). The combined organic layer (ether and CHCl₃:n-BuOH)was washed with water and a slightly yellow precipitate was formed inthe mixed solvents, at which point the solvents were evaporated undervacuum. A slightly yellow solid was formed and was recrystallized fromacetone/hexane. The solid was dried under vacuum, yield 60%.

Bis-(2(1-adamantyl)ethyl)phosphate and 11 are mixed in DMSO. EDC (3 eq),NHS (2.2 eq), and DIEA (1 eq) are added to the solution. Solution isstirred under argon for 16 hours.Bis-(2(1-adamantyl)ethyl)phosphate-Gly-CPT is precipitated with ether,washed extensively with this solvent, and dried under high vacuum. Thiscompound and Di-CD-PEG 46 are mixed in DMSO to form inclusionpolymer-CPT conjugate 48.

Example 40 Synthesis of AD-Peg-Folate 49

The following procedure is the synthesis of AD-Peg₅₀₀₀-Folate. Thismethod can be adapted for the synthesis of any Guestmolecule-Linker-Ligand tri-component molecules.

1. Synthesis of AD-Peg-NH₂

266 mg of FMOC-PEG₅₀₀₀-NHS (78.2 μmol, Shearwater Polymers, HuntsvilleAla.) were added to a glass vial equipped with a magnetic stirbar. 10eq. of 1-adamantane-methylamine (1.5 mmol, Aldrich) dissolved in 3 mL ofdichloromethane were then added and the solution stirred overnight atroom temperature. The solvent was removed in vacuo and water was addedto the remaining solution to dissolve the PEG product. The solution wascentrifuged at 20K rcf for 10 minutes, whereupon theadamantane-methylamine phase-separated as a denser liquid. The aqueousportion was collected and water removed in vacuo. The remaining viscousliquid was redissolved in 20% piperidine in DMF for FMOC deprotectionand stirred for 30 minutes at room temperature. The solvent was removedin vacuo, washed several times with DMF, redissolved in water, and runon an anionic exchange column to remove unreacted PEG. The firstfractions were collected and lyophilized to yield 222 mg of a white,fluffy powder (76% yield) of the desired product which was confirmed byMALDI-TOF analysis.

2. Synthesis of N-Hydroxysuccinimide-Folate (NHS-Folate)

NHS-folate is synthesized according to the method of Lee and Low (Lee,R. J; Low, P. S. J. Biol. Chem. 1994, 269, 3198-3204). Folic acid (5 g,11.3 mmol; Sigma) is dissolved in DMSO (100 ml) and triethylamine (2.5ml) and reacted with N-hydroxysuccinimide (2.6 g, 22.6 mmol) anddicyclohexylcarbodiimide (4.7 g, 22.7 mmol) over-night at roomtemperature. The solution is filtered and concentrated under reducedpressure. NHS-folate is precipitated using diethyl ether (yellow-orangeprecipitate), and washed 2-3 times in anhydrous ether, dried undervacuum, and stored at −20° C.

3. AD-Peg₅₀₀₀-Folate

AD-Peg5000-NH₂ and NHS-Folate are mixed at 1:1 eq. in DMSO solution.DIEA (2 eq) is added. The mixture is allowed to stir at room temperaturefor overnight. The DMSO solution is then dialyzed against 3500 MWCO(Spectra/Por 7) membrane for 48 hours. AD-Peg5000-Folate 49 is obtainedafter lyophilization.

Example 41 Formulation of the AD-Peg-Folate and a CD-Polymer-CPTConjugate

A typical procedure: A CD-polymer CPT conjugate is dissolved in a D5Wbuffer. A D5W solution containing 0.1-1 eq (mols of repeat unit) ofAD-Peg-Folate solution is added to polymer solution. Particle sizes ofpolymer are measured before and after adding of AD-Peg-Folate usinglight scattering. This solution is used for either in vitro or in vivotest for the analysis of folate targeting effects.

Example 42 Covalent Linking a Targeting Ligand (e.g., Folate) to aCD-CPT Polymer Conjugate (eg PEI-CD-GlyCPT) 50

PEI-CD-GlyCPT 44 and Folate-NHS (0.1-0.5 eq) are mixed in DMSO andstirred for 24 hours. The polymer is crashed out with ether and washedextensively with this solvent until no free folate molecule can bedetected. The resulting CD-polymer-CPT-Folate conjugate 50 is driedunder vacuum.

Example 43 Crosslinking of CD-CPT Polymer Using Ad-Peg-Ad. 51

A typical procedure: AD-Peg-AD 45 (0.01-0.1 eq of CD-polymer repeatunit) and CD-polymer CPT conjugate are mixed in a minimum amount ofDMSO. The resulting viscous solution is precipitated with ether anddried under vacuum to yield lightly crosslinked polymer 51. 51 shouldstill maintain small particle size in solution, have good watersolubility, and have higher molecular weight than parent CD-polymer CPTconjugate.

Example 44 In Vivo Tests of Camptothecin Polymer Conjugates HGGG6,LGGG10, HG6, and HGGG10 (Synthesized According to Example 28)

A. In Vivo Toxicity and Blood Chemistry Analysis from Dosing with ParentPolymer 36e

The toxicity of 36e and its effects on blood chemistry were evaluated infemale Charles River nude mice (13-14 weeks old). Four treatment groupsof six mice each were treated with a D5W solution of 36e at a dose of240 mg/kg, 160 mg/kg, 80 mg/kg or D5W alone by i.v. tail vein injectionon Days 1, 5, and 9, respectively. Dosing volume was determined basedupon a ratio of 200 μl for a 20 g mouse, and was scaled appropriatelyaccording to the actual BW of the mice. The BWs of the mice werefollowed daily for the first 5 days and then twice a week, thereafter.Blood samples (150-200 μl) were collected from each mouse byretro-orbital bleeding under isoflourane on Day 12. Samples from threemice in each group were used for complete blood count (CBC) analyses,while blood samples from the remaining three mice in each group wereprocessed for blood chemistry analyses. The study was stopped at Day 23.All mice were euthanized by cardiac puncture under CO₂, and blood fromeach mouse was collected for CBC and blood chemistry analysis in thesame manner as on Day 12.

There was no significant difference in BW loss, CBC or blood chemistrydata between any of the 36e treated groups and D5W control groupthroughout the study, and no time dependent effects were observed over23 days for all the treated groups. 36e was well tolerated by mice atthe maximum dose treated (240 mg/kg).

B. Determination of Maximum Tolerable Dose (MTD) for CDP-CPT Conjugates

The MTD was determined using female Charles River nude mice (15-16 weeksold) for HG6, LGGG10, HGGG10. A 5% (w/v) of dextrose solution (D5W) ofthe polymer-CPT conjugates was freshly prepared before each injection.Doses for the treatment groups ranged from 2.25 mg CPT/kg to 54 mgCPT/kg. Dosing was administered intravenously (i.v.) by tail veininjection on Days 1, 5, and 9. The dosing volume was determined basedupon a ratio of 200 μl for a 20 g mouse, and was scaled appropriatelyaccording to actual body weight (BW) of the mice. Three to five micewere used in each treatment group. The BWs of the mice were followeddaily for the first 5 days and then twice a week, thereafter. The MTDwas defined as the highest administered dose that resulted in a decreaseof mean group BW of less than 20% or the highest administered dose thatdid not result in death of any animal in that group. The maximum meanbody weight loss and treatment related deaths for all treated groups arelisted in Table 5.

TABLE 5 Treatment response for the MTD study. Nude mice (n = 3-6) weretreated i.v. by tail vein injection. Max % BW loss; Agent mg/kg^(a)Day^(b) N_(TR)/N^(c) D5W — −2.5%; Day 3 0/6 36e 240 −2.0%; Day 3 0/6 36e160 −3.5%; Day 13 0/6 36e 80 −2.3%; Day 3 0/6 LGGG10 54 −20.6%; Day 33/3 LGGG10 36 −9.3%; Day 13 0/3 LGGG10 18 0 0/3 LGGG10 9 0 0/5 LGGG104.5 −0.8%; Day 13 0/5 HG6 54 −28.5%; Day 3 3/3 HG6 36 −23.9%; Day 3 3/3HG6 18 −22.1%; Day 3 3/3 HG6 9 −6.1%; Day 9 0/5 HG6 4.5 −4.4%; Day 5 0/5HG6 2.25 −2.9%; Day 9 0/5 HGGG10 54 — 3/3 HGGG10 36 −34%; Day 5 3/3HGGG10 18 −16%; Day 3 1/3 HGGG10 9 −3.3%; Day 9 0/5 HGGG10 4.5 −2.5%;Day 9 0/5 ^(a)Mg CDP/kg for the CDP polymer and mg CPT/kg for the threeconjugates tested. ^(b)Maximum body weight (BW) loss observed postinjection ^(c)Number of treatment-related deaths (N_(TR)) to the numberof mice treated (N)

The MTD of LGGG10, HG6, and HGGG10 were determined to be 36 mg CPT/kg, 9mg CPT/kg, and 9 mg CPT/kg, respectively. Based on the structuralsimilarities between HGGG6 and HGGG10, it is expected that the MTD forthese two groups are similar. Therefore, the MTD of HGGG6 (not tested)was assumed to be 9 mg CPT/kg.

C. Antitumor Efficacy Study

The antitumor efficacy study was performed using female Charles Rivernude mice (15-16 weeks old). A fragment (1 mm³) of human LS174T coloncarcinoma tissue was implanted subcutaneously (s.c.) into the rightflank of each test mouse approximately 14-18 days before dosing. Thetumor volume was determined by measuring the tumor in two dimensionswith calipers and calculated using the formula: tumorvolume=(length×width²)/2. Tumor volume was converted to tumor weightassuming 1 mm³ is equal to 1 mg tumor in weight. Treatment wasinitialized when mean tumor size reached approximately 60-100 mg (Day1). The animals were sorted into twelve groups. Each group consisted ofseven mice with tumor sizes ranging from 62.5-144.0 mg with group meantumor sizes of 88.6-90.7 mg. Mice in each group were treated accordingto the protocol listed in Table 6. All conjugate treatments wereadministered intravenously by tail vein injection. Tumor sizes weremeasured twice a week for the duration of the experiment. At the end ofstudy, tumors from each euthanized mouse were harvested and frozen at−80° C.

TABLE 6 Dosing protocol for efficacy study.^(a) Dose (mg Group AgentCPT/kg)^(b) Route^(c) Schedule^(d) 1 D5W — i.v. Q4D x 3 2 CPT  9 i.p.Q4D x 2^(e) 3 Irinotecan 100^(f) i.p. Qwk x 3 4 HGGG6  9 i.v. Q4D x 3 5HGGG6  4.5 i.v. Q4D x 3 6 LGGG10  36 i.v. Q4D x 3 7 LGGG10  18 i.v. Q4Dx 3 8 LGGG10  9 i.v. Q4D x 3 9 HG6  9 i.v. Q4D x 3 10 HG6  4.5 i.v. Q4Dx 3 11 HGGG10  9 i.v. Q4D x 3 12 HGGG10  4.5 i.v. Q4D x 3 ^(a)Seven micewere used in each group ^(b)Doses are equivalent of CPT except for group3 ^(c)i.p. = intraperitoneal, i.v. = intravenous ^(d)Administrationschedules were abbreviated as: Q4D x 3 = three injection with four-dayintervals, Qwk x 3 = three injection with one-week interval, first dosewas initialized on day 1 for all groups. ^(e)The scheduled third dosewas not given due to the emerging toxicity ^(f)100 mg irinotecan/kg

Each animal was euthanized when tumor weight reached the predeterminedendpoint size (1,500 mg). The time-to-endpoint (TTE) for each mouse wascalculated from the equation: TTE=(log(endpoint-b))/m, where b and m arethe intercept and the slope, respectively, of the line obtained bylinear regression of a log-transformed tumor growth data set comprisedof the first observation that exceeded the study endpoint volume and thethree consecutive observations that immediately preceded the attainmentof the endpoint volume. Animals that do not reach the endpoint wereassigned a TTE value equal to the last day of the study (114 days).Animals classified as treatment-related deaths (TR) were assigned a TTEvalue equal to the day of death. Animals classified asnon-treatment-related death (NTR) are excluded from TTE calculations.Tumor growth delay (TGD), defined as the increase in the median time toendpoint (TTE) in a treatment group compared to the control group, wasone parameter investigated to evaluate treatment efficacy. TGD iscalculated as the difference between the median TTE for a treatmentgroup and the median TTE of the control group (TGD=T−C) and is expressedin days, and as a percentage of the median TTE of the control group; %TGD=(T−C)/C where T is equal to median TTE for a treatment groups and Cis equal to median TTE for the control, Group 1.

Toxicity. Animals were weighed daily on Days 1-5, then twice weeklythereafter. Mice were examined frequently for overt signs of anyadverse, drug-related side effects. Acceptable toxicity for cancer drugsin mice is defined by the NCI as a group mean body-weight loss of lessthan 20% during the study, and not more than one toxic death among seventreated animals.

Results for this efficacy study that include median TTE values, mediantumor burden on day 114, treatment response and deaths are summarized inTable 7.

TABLE 7 Median Tumor N_(TR) ^(e)/ Median % Burden in N_(NTR) ^(f)/ P vsP vs Group TTE^(a) T − C^(b) TGD^(c) mg (N_(s) ^(d)) N_(EU) ^(g) D5W^(h)CPT^(i) 1 34.9 — — — (0) 0/1/6 — — 2 51.4 16.5  47% — (0) 2/0/5 0.2128 —3 68.7 33.8  97% 1152 (3) 0/0/4 0.0002 0.0253 4 114.0 79.1 227% 256 (5)1/0/1 0.0040 0.0115 5 65.6 30.7  88% 566 (2) 0/1/4 0.0046 0.1369 6 100.065.1 187% 666 (3) 4/0/0 0.0272 0.0289 7 75.6 40.7 117% 221 (3) 0/0/40.0018 0.0601 8 63.2 28.3  81% 700 (1) 1/0/5 0.0006 0.1064 9 114.0 79.1227% 394 (4) 0/0/3 0.0002 0.0028 10 74.2 39.3 113% 668 (2) 1/1/3 0.00160.0673 11 114.0 79.1 227% 500 (5) 1/0/1 0.0040 0.0050 12 78.0 43.1 123%1010 (2) 0/0/6 0.0006 0.0392 ^(a)TTE = Time (Days) to endpoint (1500 mg)^(b)T − C = Difference between TTE (Days) of treated versus controlgroup ^(c)% TGD = [(T − C)/C] ^(d)Mice surviving ^(e)N_(TR) = Number oftreatment-related death ^(f)N_(NTR) = Number of non-treatment-relateddeath ^(g)N_(EU) = Number of mice euthanized after reaching endpoint^(h)P value versus the D5W treatment group (Group 1) ^(i)P value versusthe CPT treatment group (Group 2)

One NTR death on day 72 was observed in the control animals treated withD5W. Tumors in the other six control mice grew to the 1500 mg endpointsize, yielding a median TTE of 34.9 days (Table 7).

Two treatment-related deaths were reported on Day 9 for CPT at 9 mg/kg.Thus, CPT must be considered to be toxic at this dose in thisexperiment. The median TTE for this group was 51.4 days, correspondingto a 16.5 day T−C and a 47% TGD, relative to untreated control mice (notsignificant). No animal in Group 2 survived to Day 114.

Group 3 received irinotecan i.p. at 100 mg/kg (Qwk×3). The median TTEfor Group 3 was 68.7 days, corresponding to a significant 33.8 day T−Cand a 97% TGD, relative to control mice (P<0.01). Three animals survivedto Day 114 with a median tumor burden of 1,152 mg. No regressions wererecorded.

Groups 4 and 5 received HGGG6 i.v. Q4D×3 at 9 and 4.5 mg CPT/kg,respectively. One treatment-related death was observed on Day 16 inGroup 4, and one NTR death was recorded on Day 37 in Group 5. The medianTTE for Group 4 was 114 days, the maximum possible value in this study.This TTE value corresponds to a significant 79.1 day T−C and a 227% TGD,relative to control (P<0.01). Tumors in five mice of Group 4 did notreach the 1,500 mg endpoint. These five mice had a median tumor burdenof 256 mg on Day 114. The median TTE for Group 5 was 65.6 days, andcorresponds to a significant 30.7 day T−C and an 88% TGD, relative tocontrol (P<0.01).

Groups 6-8 were treated with LGGG101.v. Q4D×3 at 36, 18, and 9 mgCPT/kg, respectively. Although no death was observed in MTD study usingthis conjugate in non-tumor bearing mice at 36 mg CPT/kg (Table 5), fourtreatment-related deaths were recorded in Group 6 when tumor-bearingmice were given at this dose, two on Day 16 and one each on Days 75 and100. These results indicate that 36 mg CPT/kg is probably over the MTDof LGGG10. As shown in Table 5, no significant body weight loss wasrecorded in the MTD study when the mice were dosed at 18 mg CPT/kg,indicating that this dose is below the MTD. Therefore, the MTD of LGGG10lies somewhere between 18 to 36 mg CPT/kg. The median TTE for Group 7(18 mg CPT/kg) was 75.6 days. This

TTE value corresponds to a significant 40.7 day T−C and a 117% TGD,relative to control mice (P<0.01). Three mice in this group had a mediantumor burden of 221 mg on Day 114. One late TR death was recorded on Day103 in Group 8 (9 mg CPT/kg). The median TTE for Group 8 was 63.2 days.This TTE value corresponds to a significant 28.3 day T−C and an 81% TGD,relative to untreated control mice (P<0.01). The remaining mouse in thisgroup had a tumor burden of 700 mg on Day 114.

Groups 9 and 10 were dosed with HG6 i.v. Q4D×3 at 9 and 4.5 mg CPT/kg,respectively. One TR and one NTR death were recorded in Group 10 on Days47 and 84, respectively. The median TTE for Group 9 was the maximum, 114days. This TTE value corresponds to a significant 79.1 day T−C and a227% TGD, relative to untreated control mice (P<0.01). Four mice inGroup 9 had a median tumor burden of 394 mg on Day 114. The median TTEfor Group 10 was 74.2 days. This TTE value corresponds to a significant39.3 day T−C and a 113% TGD, relative to control mice (P<0.01). Theremaining two mice in Group 10 had a median tumor burden of 668 mg onDay 114.

Groups 11 and 12 were dosed with HGGG10i.v. Q4D×3 at 9 and 4.5 mgCPT/kg, respectively. One treatment-related death was recorded on Day 16in Group 11. The median TTE for Groups 11 and 12 were 114 days and 78days, respectively. The TTE value for Group 11 corresponds to asignificant 79.1 day T−C and a 227% TGD, relative to control mice(P<0.01). Tumors in five mice in Group 11 did not reach the endpoint;these five mice had a median tumor burden of 500 mg on Day 114. The TTEvalue of Group 12 corresponds to a significant 43.1 day T−C and a 123%TGD, relative to control mice (P<0.01). The remaining two mice in thisgroup had a median tumor burden of 1,010 mg on Day 114.

The tumor growth curve as a function of time for the D5W, CPT,irinotecan, LGGG10 at its highest non-toxic dose tested (18 mg CPT/kg),and the other three conjugates with high MW polymer (HGGG6, HG6, HGGG10)at their MTDs are shown in FIG. 8. The three high MW conjugatesadministered at their MTDs displayed more prolonged tumor growthinhibition compared to D5W, CPT and irinotecan. The median tumor growthcurves for HGGG6, HG6 and HGGG10 that are illustrated in FIG. 9 showthat there is a distinct dose response for all three of these polymerswhen dosed as their MTD and at half of their MTD. The medium tumorgrowth curves for LGGG10 and HGGG10 each dosed at 9 mg CPT/kg asillustrated in FIG. 10 demonstrate that high MW polymer-drug conjugatehas greater antitumor effect when compared to the low MW conjugatespresumably due to the enhanced accumulation (EPR effect) and reducedrenal clearance.

Mean Body Weight Loss of Mice. Mean body weight (MBW) losses as afunction of time are plotted for D5W, CPT, irinotecan and the threeconjugates containing high MW polymer at their MTDs (FIG. 11). MaximumMBW losses observed in Group 2 (CPT) and the two conjugates with thetriglycine linker dosed at their MTDs (Groups 4 and 11) were 13.1%,18.3%, and 12.6%, respectively. Maximum MBW loss of HG6 (3.4%), the onlyconjugate with a glycine linker, was similar to the maximum MBW lossrecorded for irinotecan (5.0%). Negligible (<5%) maximum group meanbody-weight losses were recorded in all the other treatment groups andin the D5W group. Mean body weight returned to baseline levels for alltreatment groups following cessation of therapy.

D Correlation of Tumor Size of Enthanized Mouse and the CPTConcentration in Corresponding Tumor.

Each tumor harvested from mice at the completion of the LS 174txenograft mouse study was thawed and placed in a 2 ml lysis tube (LysingMatrix D, Qbiogen). 300 μL of lysis reagent (Cellytic—MT MommalianTissue Lysis/Extraction reagent) was added to each tube. The tissue washomogenized on a FastPrep FP12 homogenizer (Qbiogen) at 5 m/s for 40sec. Homogenization was repeated six times with a 10 min intervalbetween successive homogenization. The homogenized solution wascentrifuged at 14000 g for 15 min at 10° C. 90 μL of the solution wassyringed out to which 10 μL 1N NaOH was added. An aliquot of 400 μL MeOHwas added to this solution after allowing the homogenized solution tostand for 2 h at room temperature. The solution was centrifuged for 15min at 14000 g. The supernatant (270 μL) was mixed with 30 μL 1N HCl andinjected into an HPLC for analysis. The correlation of CPT concentration(ng/mg tissue) to tumor size (in mg) is illustrated in FIG. 12. CPTconcentration was inversely correlated to tumor size.

Example 45 Synthesis of Poly(CDDC-PEG)-Amphotericin B 52 Via AmideLinker

Poly(CDDC-PEG) (788 mg) and 1,1′-carbonyl diimidazole (CDI, 1.45 g, 50eq.) were stirred in anhydrous DMSO (10 mL) in the presence of DMAP (429mg, 20 eq) for 16 h. Ether (200 mL) was added to the mixture toprecipitate poly(CDDC-PEG)-carbonyl-imidazole. The resulting yellowsolid was washed with ether 2×200 mL and dried under vacuum. The solidwas dissolved in anhydrous DMSO (15 mL), followed by adding AmB (332 mg,2 eq) and DMAP (43.0 mg, 2 eq). The solution was stirred in dark for 48h and dialyzed in water using 25000 MWCO membrane for 3 days. Thesolution was then filtered using 0.2 μm filter and lyophilized. A yellowsolid (920 mg) 52 was obtained. The wt % of AmB is around 13%.

Example 46 Synthesis of Poly(CDDC-PEG)-Amphotericin B 53 Via ImineLinker

3 and PEG-DiSPA (1:1 ratio) were dried under vacuum at room temperature.DMSO (10 mg of 3/mL DMSO) was added to the solid and followed by addingof DIEA (2 eq) to the mixture. Polymer was crashed out with excess ether5 days later and dialyzed using 25000 MWCO membrane for 48 h. The yieldof poly(CDDC-PEG) is 80-95%. The Mw of polymer was determined using GPCto be 70-100 kDa.

Poly(CDDC-PEG) (1.124 g, 0.25 mmol) was dissolved in water (55 mL).NaIO₄ (0.264 g, 5 eq.) was added. The solution was stirred in dark atroom temperature for 20 min and stored at 4° C. for 24 h in dark. BaCl₂solution was added (5.05 eq) to the solution to give immediateprecipitation of Ba(IO₄)₂. The precipitate was filtered. SaturatedNa₂CO₃ solution was added to adjust pH to 11. Amphotericin B (343 mg,1.5 eq) was then added to solution and stirred at rt in dark for 48 h.The pH of solution was maintained to be 11 by adding NaOH (0.1N)throughout the reaction. The solution was dialyzed at 4° C. for 48 husing 25000 MWCO and lyophilized to give 1.03 g polymer-AmB conjugate 53as a yellow powder. The wt % of AmB is determined to be 18 using UVspectrometer at 405 nm.

D. References

Additional cyclodextrin-containing polymers that can be modifiedaccording to the teachings of the present invention, as well as methodsof preparing such polymers, are disclosed in U.S. patent applicationSer. Nos. 09/203,556, 09/339,818, 09/453,707, 10/021,294, and10/021,312, all of which are hereby incorporated herein by reference intheir entireties.

All of the references, patents, and publications cited herein are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

We claim:
 1. A pharmaceutical composition comprising a sugar and a watersoluble linear polymer conjugate, wherein the water soluble linearpolymer conjugate comprises: a linear polymer comprising betacyclodextrin moieties and comonomers which do not contain betacyclodextrin moieties (comonomers); and taxane moieties covalentlylinked to the linear polymer via glycine linkers, wherein the taxanemoieties are cleaved from the water soluble linear polymer conjugateunder biological conditions to release the taxane moieties; wherein thewater soluble linear polymer conjugate comprises at least four betacyclodextrin moieties and at least four comonomers.
 2. Thepharmaceutical composition of claim 1, wherein the comonomers comprisespolyethylene glycol.
 3. The pharmaceutical composition of claim 2,wherein the polyethylene glycol has a molecular weight of 0.2 kDa to 5kD.
 4. The pharmaceutical composition of claim 1, wherein each of the atleast four beta cyclodextrin moieties alternates with each of the atleast four comonomers in the water soluble linear polymer conjugate. 5.The pharmaceutical composition of claim 1, wherein the taxane moietiesare at least 5% by weight of the water soluble linear polymer conjugate.6. The pharmaceutical composition of claim 1, wherein administration ofthe composition to a patient results in release of the taxane moietiesover a period of at least 6 hours.
 7. The pharmaceutical composition ofclaim 1, wherein administration of the composition to a patient resultsin release of the taxane moieties over a period of 6 hours to a month.8. The pharmaceutical composition of claim 1, wherein, uponadministration of the composition to a patient, the rate of the taxanemoieties release is dependent primarily upon the rate of hydrolysis asopposed to enzymatic cleavage.
 9. The pharmaceutical composition ofclaim 1, wherein the water soluble linear polymer conjugate has amolecular weight of 10,000-500,000 amu.
 10. The pharmaceuticalcomposition of claim 1, wherein the water soluble linear polymerconjugate has a molecular weight of 30,000-520,000 amu.
 11. Thepharmaceutical composition of claim 1, wherein the water soluble linearpolymer conjugate has a molecular weight of 70,000-150,000 amu.
 12. Thepharmaceutical composition of claim 1, wherein the beta cyclodextrinmoieties are at least about 10% by weight of the linear polymer.
 13. Thepharmaceutical composition of claim 1, wherein the beta cyclodextrinmoieties are at least about 20% by weight of the linear polymer.
 14. Thepharmaceutical composition of claim 1, wherein the beta cyclodextrinmoieties are at least about 30% by weight of the linear polymer.
 15. Thepharmaceutical composition of claim 1, further comprising apharmaceutically acceptable carrier.
 16. A pharmaceutical compositioncomprising a sugar and a water soluble linear polymer conjugatecomprising: a linear polymer comprising beta cyclodextrin moieties andcomonomers comprising a polyethylene glycol; and taxane moietiescovalently linked to the linear polymer via glycine linkers, wherein thetaxane moieties are cleaved from the water soluble linear polymerconjugate under biological conditions to release the taxane moieties;wherein the water soluble linear polymer conjugate comprises at leastfour beta cyclodextrin moieties and at least four comonomers.
 17. Thepharmaceutical composition of claim 16, further comprising apharmaceutically acceptable carrier.
 18. The pharmaceutical compositionof claim 16, wherein the water soluble linear polymer conjugate has amolecular weight of 30,000-520,000 amu.
 19. The pharmaceuticalcomposition of claim 16, wherein the water soluble linear polymerconjugate has a molecular weight of 70,000-150,000 amu.
 20. Thepharmaceutical composition of claim 16, wherein the taxane moieties areat least 5% by weight of the water soluble linear polymer conjugate. 21.The pharmaceutical composition of claim 16, wherein administration ofthe composition to a patient results in release of the taxane moietiesover a period of at least 6 hours.
 22. A pharmaceutical compositioncomprising a sugar and a water soluble linear polymer conjugatecomprising: a linear polymer comprising beta cyclodextrin moieties andcomonomers which do not contain beta cyclodextrin moieties (comonomers);and taxane moieties covalently linked to the linear polymer via glycinelinkers, wherein the taxane moieties are cleaved from the water solublelinear polymer conjugate under biological conditions to release thetaxane moieties; wherein the water soluble linear polymer conjugatecomprises at least four beta cyclodextrin moieties and at least fourcomonomers and has a molecular weight of 30,000-520,000 amu.
 23. Thepharmaceutical composition of claim 22, further comprising apharmaceutically acceptable carrier.
 24. The pharmaceutical compositionof claim 22, wherein the taxane moieties are at least 5% by weight ofthe water soluble linear polymer conjugate.
 25. The pharmaceuticalcomposition of claim 22, wherein administration of the composition to apatient results in release of the taxane moieties over a period of atleast 6 hours.
 26. The pharmaceutical composition of claim 22, having amolecular weight of 70,000-150,000 amu.
 27. The pharmaceuticalcomposition of claim 22, wherein each of the comonomers comprises apolyethylene glycol.
 28. The pharmaceutical composition of claim 1,wherein the sugar is mannitol.
 29. The pharmaceutical composition ofclaim 16, wherein the sugar is mannitol.
 30. The pharmaceuticalcomposition of claim 22, wherein the sugar is mannitol.
 31. Thepharmaceutical composition of claim 3, wherein the sugar is mannitol.32. The pharmaceutical composition of claim 4, wherein the sugar ismannitol.
 33. The pharmaceutical composition of claim 24, wherein thesugar is mannitol.
 34. The pharmaceutical composition of claim 27,wherein the sugar is mannitol.
 35. The pharmaceutical composition ofclaim 16, wherein the beta cyclodextrin moieties are at least about 10%by weight of the linear polymer.
 36. The pharmaceutical composition ofclaim 16, wherein the beta cyclodextrin moieties are at least about 20%by weight of the linear polymer.
 37. The pharmaceutical composition ofclaim 16, wherein the beta cyclodextrin moieties are at least about 30%by weight of the linear polymer.
 38. The pharmaceutical composition ofclaim 22, wherein the beta cyclodextrin moieties are at least about 10%by weight of the linear polymer.
 39. The pharmaceutical composition ofclaim 22, wherein the beta cyclodextrin moieties are at least about 20%by weight of the linear polymer.
 40. The pharmaceutical composition ofclaim 22, wherein the beta cyclodextrin moieties are at least about 30%by weight of the linear polymer.
 41. The pharmaceutical composition ofclaim 16, wherein each of the at least four beta cyclodextrin moietiesalternates with each of the at least four comonomers in the watersoluble linear polymer conjugate, and the sugar is mannitol.
 42. Thepharmaceutical composition of claim 41, wherein each of the at leastfour comonomers comprises polyethylene glycol having a molecular weightof 0.2 kDa to 5 kDa.